COPY-UP MUTANTS OF THE PLASMID RK2 REPLICATION INITIATION PROTEIN AREDEFECTIVE IN COUPLING RK2 REPLICATION ORIGINS

The broad host range plasmid RK2 replicates and regulates its copy num ber in a wide range of Gram-negative bacteria, The plasmid-encoded tra ns-acting replication protein TrfA and the origin of replication oriV are sufficient for controlled replication of the plasmid in all Gram-n egative bacteria tested, The TrfA protein binds specifically to direct repeat sequences (iterons) at the origin of replication, A replicatio n control model, designated handcuffing or coupling, has been proposed whereby the formation of coupled TrfA-oriV complexes between plasmid molecules results in hindrance of origin activity and, consequently, a shut-down of plasmid replication under conditions of higher than norm al copy number, Therefore, according to this model, the coupling activ ity of an initiation protein is essential for copy number control and a copy-up initiation protein mutant should have reduced ability to for m coupled complexes. To test this model for plasmid RK2, two previousl y characterized copy-up TrfA mutations, trfA-254D and trfA-267L, were combined and the resulting copy-up double mutant TrfA protein TrfA-254 D/267L was characterized, Despite initiating runaway (uncontrolled) re plication in vivo, the copy-up double-mutant TrfA protein exhibited re plication kinetics similar to the wild-type protein in vitro. Purified TrfA-254D, TrfA-267L, and TrfA-254D/267L proteins were then examined for binding to the iterons and for coupling activity using an in vitro ligase-catalyzed multimerization assay, It was found that both single and double TrfA mutant proteins exhibited substantially reduced (sing le mutants) or barely detectable (double mutant) levels of coupling ac tivity while not being diminished in their capacity to bind to the ori gin of replication, These observations provide direct evidence in supp ort of the coupling model of replication control.