DnaA-mediated regulation of phage λ-derived replicons in the absence of pR and Cro function

Bacteriophage A-derived replicons can replicate in Escherichia coli cells as plasmids. In the control of replication of these plasmids, an important role was ascribed to the A Cro repressor autoregulatory loop. However, the o(R)/p(R)-cro-t(R1)-cll' region could be replaced by the p(tetA) promoter under the control of the TetR repressor, producing plasmid pTC lambda 1. Here, we demonstrate that stable maintenance of pTC lambda 1 depends on the host DnaA function because deletion of one of DnaA-binding sequences present in pTC lambda 1 resulted in a decrease in the plasmid (pTC lambda 2) copy number and poor maintenance of pTC lambda 2 in E. coli. Moreover, in contrast to the replication of the wild-type lambda plasmid, previously found to be positively regulated by DnaA (acting on a relaxed DnaA box situated immediately downstream of the p(R) promoter), the replication of pTC plasmids (devoid of p(R)) was found to be negatively regulated by DnaA. Contrary to wild-type lambda plasmids, in cells harboring lambda cro [temperature-sensitive (ts)] or pTC lambda 1 (but not pTC lambda 2) plasmid, the lambda replication complex was heat shock resistant; this complex, however, disassembled after inactivation of DnaA function. This disassembly was blocked by DNA gyrase inhibitors. According to our model outlined previously, we propose that the heat shock resistance of the replication complex of lambda cro(-) plasmids depends on the interaction of the DNA-bound DnaA protein with the DNA-bound lambda replication complex. The replication complex-DnaA-lambda DNA structure may be directly related to the role of DnaA as the Cro-replacing negative regulator of lambda cro(-) plasmid replication. (C) 1998 Academic Press.