A TRANSCRIPTION TERMINATOR SIGNAL NECESSARY FOR PLASMID COLLB-P9 REPLICATION

Replication of the Incl alpha plasmid Collb-P9 requires the repZ gene, which encodes an essential, unstable initiator protein termed RepZ. A lthough many functional features of the Collb-P9 replicon resemble tho se of structurally unrelated IncFll plasmids R1 and NR1, the role of t ranscription of repZ towards the replication origin is poorly understo od. Using a series of deletion and substitution mutants of the Collb-P 9 replicon, we found that RepZ prefers to act in cis and that a spacer sequence between repZ and the origin is required for replication. Thi s spacer element, referred to as CIS, retained strong transcription te rminator activity. Efficient transcription terminators, whether Rho-de pendent or -independent, were capable of replacing CIS function for in vivo replication; Collb-P9 replicated better as transcription termina ted more efficiently within CIS. When the CIS element was substituted for by a strong Rho-dependent terminator, such as lambda tR1 or E. col i trp t', in vivo replication of these recombinant replicons became de pendent on the Rho factor, in contrast to the authentic Collb-P9 repli con.