ANALYSIS OF THE MULTIMER RESOLUTION SYSTEM ENCODED BY THE PARCBA OPERON OF BROAD-HOST-RANGE PLASMID RP4

The broad-host-range plasmid RP4 encodes a highly efficient partitioni ng function, termed par, that is capable of stabilizing plasmids in a variety of Gram-negative bacteria independently of the nature of the r eplicon. The mechanism responsible for plasmid stabilization by this l ocus appears to be a complex system which includes a site-specific rec ombination system mediating resolution of plasmid multimers. In this r eport we present a detailed study on this multimer resolution system ( mrs). The parA gene encodes two forms of a resolvase capable of cataly sing site-specific recombination between specific sites situated in th e promoter region of the parCBA operon. The two ParA proteins that are produced as a result of independent translation initiation at two dif ferent start codons within the same open reading frame were overexpres sed in Escherichia coli and partially purified. Both forms of the enzy me are able to recombine a supercoiled cointegrate substrate containin g two cis-acting elements with the same orientation in an in vitro res olution assay. ParA-mediated, site-specific recombination was found to be independent of any other gene product encoded by the RP4 par locus in vitro and in vivo. The DNA-binding sites for the ParA resolvase we re determined using DNase I protection experiments. The results identi fied three binding sites within the mrs cis-acting region. Both the bi ochemical properties of the ParA protein and the organization of the c is-acting recombination site revealed a high degree of similarity to t he site-specific recombination systems of Tn3-like transposable elemen ts suggesting an evolutionary relationship.