The long-term goal of this project is to characterize at the molecular level the DNA processing steps required for the conjugal transfer of broad host-range plasmids. The proposed work will be focussed on the multi-copy plasmid R1162, which encodes three proteins (MobA-C) required for its conjugal mobilization. Underlying biochemical similarities between initiation of transfer (nicking of duplex DNA) and termination (ligation of a single, linear DNA strand) will be identified and characterized. How MobA and MobC bring about the localized melting of oriT DNA in the relaxosome will be investigated. The carboxy-terminal region of MobA is a primase, also synthesized separately, that is required for the vegetative replication of R1162. The role of the primase domain in complementary strand synthesis after transfer will be examined. The function of MobB protein in conjugal mobilization is presently unknown. The possibility will be explored that this protein regulates the level of nicked molecules available to initiate a round of transfer by disrupting the association between oriT DNA and MobA protein. Purified MobA protein can cleave and ligate single-stranded oriT DNA in vitro. The mechanism of this reaction, and its compatibility with a model for the termination of transfer, will be investigated. Experimental approaches in this study will include isolating second-site suppressor mutations, chemical probing of the relaxosome, and reconstituting MobA oriT DNA interactions in vitro with purified components.
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