The broad host-range plasmid R1162, widely disseminated among different Gram-negative bacterial species, is conjugally transferred from cell to cell by other, self-transmissible plasmids. Mobilization requires the formation of a relaxosome comprised of a plasmid origin of transfer (oriT), bound to three, plasmid-encoded mobilization (Mob) proteins. This complex is required both to process the DNA for transfer, and to recognize the conjugal machinery of the transferring vector. The principal component of the relaxosome, MobA, is a DNA endonuclease and ligase. The protein domain that binds oriT will be identified by phage display, and the DNA recognition sequence determined by binding MobA to a degenerate oligonucleotide. Within the relaxosome, there is partial separation of the oriT DNA strands. How this occurs will be investigated by characterizing the interaction of MobA and the other relaxosome proteins with oriT heteroduplexes. Genetic evidence indicates that MobB stabilizes the relaxosome and promotes efficient transfer by interacting with MobA. Physical evidence for this interaction will be obtained by coimmunoprecipitation. The conjugal apparatus of the IncP-1 plasmid RK2 efficiently mobilizes R1162. The protein component recognized by R1162 and required for the plasmid to enter a round of transfer will be identified either by an affinity overlay procedure, or by direct immunoprecipitation. The interacting domain of this protein will be mapped by phage display or by a bacterial two-hybrid assay. A co-reversion study will be carried out to establish whether this domain is also recognized by RK2 itself. In addition, mutations in R1162 that increase the frequency of mobilization will be isolated and tested for interference of RK2 cotransfer. The mob systems of plasmids R1162 and pSC101 are clearly related, with similar oriTs and MobA proteins. However, pSCl0l encodes no homologs to the R1162 proteins MobB and MobC. How pSCl0l has become independent of these proteins will investigated. Overall, these studies will provide insight into how the relaxosome directs plasmid DNA to the conjugal pore for transfer.
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