PROJECT SUMMARY Conjugation systems are widely known for their roles in disseminating mobile genetic elements (MGEs) and cargoes of antibiotic resistance, fitness, and virulence genes among many species of bacteria. The enormous medical problem associated with MGE-transmitted antibiotic resistance is enhanced by the fact that conjugation systems also code for surface adhesins or conjugative pili that promote intercellular contacts favoring transmission and establishment of robust, antibiotic-resistant biofilm communities. Furthermore, various environmental stresses, including antibiotics, have been shown to stimulate MGE transmission whereas, conversely, MGE transfer induces the SOS response and activates movement of integrons, which are reservoirs of antibiotic resistance genes. Adding to the myriad of ways conjugation can impact development of antibiotic resistance and genome evolution, we have shown that a consequence of conjugation-induced SOS is the elevated accumulation of mutations in recipient cells. To mitigate the deleterious consequences of mating-induced mutation, here termed MIM, many conjugative elements carry genes such as psiB and ssb whose products suppress the SOS response. In our studies of a model F plasmid, pED208, we discovered that PsiB and SSB are delivered through the conjugation channel, also known as a type IV secretion system (T4SS), to recipient cells. Furthermore, translocated PsiB and SSB suppress both the SOS response and MIM in transconjugants. ssb and psiB genes are carried by the maintenance/leading regions (MLRs) of F plasmids and many other conjugative plasmids, as are several other genes whose products are predicted to promote establishment of the MGE following transfer to recipient cells By use of a robust and quantitative Cre Recombinase Assay for Translocation (CRAfT), we have shown that pED208 conjugatively transfers at least 9 MLR-encoded proteins, as well as 7 chromosomally-encoded proteins with predicted or known functions in DNA metabolism and stress mitigation. In Aim 1, we will define the functional interplay of PsiB, SSB, and a third protein, PsiA, in modulating the SOS response and MIM in donor and recipient cells during conjugation. We will also evaluate the broader biological roles of PsiB and SSB translocation through studies of other model conjugation systems and intra- and interspecies matings, and by assessing effects of exogenous stresses on MGE transfer and PsiB/SSB mitigation of stress responses and mutation. In Aim 2, we will identify and characterize the biological functions of other translocated MLR proteins encoded by the model F plasmid, pED208. We will define the repertoire of chromosomally-encoded translocated proteins, and initiate studies exploring biological functions focusing initially on testing a hypothesis that mating induces the oxidative stress response and translocated SodA mitigates this stress response. Results of these studies will significantly expand our understandin...