Plasmids of incompatibility group P (IncP) are known for their extraordinary host range among Gram-negative bacteria. We have been studying one such plasmid, RK2, to learn how it controls its replication in such a wide variety of bacterial species. RK2 has an essential determinant, trfA, whose functions are in some way required for initiation of DNA replication at oriV, the RK2 replication origin. We have found that trfA gene expression is regulated. The RK2 genes korA and korB have a negative effect on trfA expression, whereas kilB1 has a positive effect. The studies outlined in this proposal are directed towards answering two major questions concerning trfA: (1) what are the mechanisms responsible for the observed regulation of trfA gene expression; and (2) what are the functions of its multiple gene products? To understand trfA regulation, we will identify important features of trfA expression at the levels of transcription, RNA processing, and translation; and examine each of these with respect to korA and korB inhibition and kilB1 enhancement of trfA expression. To determine the function of trfA, we will overproduce and purify the three trfA polypeptide products; characterize them with respect to DNA binding properties and interactions with plasmid and host proteins; place each of the trfA genes under control of a regulated promoter and use the fusions to identify which trfA product will activate oriV-specific replication, inhibit replication, and cause host cell death when overproduced. We will use this latter phenotype to select mutants in the trfA genes or in host cell genes to identify host products which interact with trfA. In addition we will begin studies on the IncP plasmid-dependent bacteriophages PRD1 and Pf3 to identify other strategies for efficient expression of essential genes in different bacterial hosts.
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