TrfA dimers play a role in copy-number control of RK2 replication

Copy-number regulation of the broad-host-range plasmid RK2 is dependent on the plasmid-encoded initiator protein, TrfA, and the RK2 origin of replicat ion. The handcuffing model for copy-number control proposes that TrfA-bound ol is reversibly couple to prevent the further initiation of plasmid repli cation when the copy number in vivo is at or above the replicon-specific co py number. TrfA mutants have been isolated which allow for oriV replication at elevated copy numbers. To better understand the mechanism of 'handcuffi ng', the copy-up TrfA(G254D/S267L) mutant was characterized further. In the present study we show by size exclusion chromatography and native gel elec trophoresis that unlike wt TrfA which is largely dimeric, purified His6-Trf A(G254D/S267L) is primarily monomeric. In vivo, TrfA33(G254D/S267L) support s replication of an RK2 ori plasmid in trans at a greatly elevated copy num ber, while in cis the plasmid exhibits runaway replication. However, expres sion of either of two previously isolated DNA-binding defective TrfA mutant s, TrfA33(P151S) or TrfA33(S257F), in a cell transformed with a mini-RK2 re plicon encoding TrfA33(G254D/S267L) results in suppression of the runaway p henotype. His6-TrfA(P151S) and His6-TrfA(S257F) purify as dimers, and when expressed in vivo are incapable of supporting RK2 plasmid replication. In c ontrast, combination of the trfA(P151S) or trfA(S257F) mutation with the tr fA(G254D/S267L) mutations results in the expression of mutant TrfA proteins which are mainly monomers and which can no longer restore copy control to replication directed by TrfA33(G254D/S267L) in vivo. On the basis of these findings a handcuffing model is proposed, whereby oriV-bound TrfA monomers are coupled by dimeric TrfA molecules. (C) 1998 Elsevier Science B.V. All r ights reserved.