DnaA-mediated regulation of phage λ-derived replicons in the absence of p(R) and Cro function

Abstract

Bacteriophage λ-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 λ 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 pTCλ1. Here, we demonstrate that stable maintenance of pTCλ1 depends on the host DnaA function because deletion of one of DnaA-binding sequences present in pTCλ1 resulted in a decrease in the plasmid (pTCλ2) copy number and poor maintenance of pTCλ2 in E. coli. Moreover, in contrast to the replication of the wild-type λ 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 λ plasmids, in cells harboring λ cro [temperature-sensitive (ts)] or pTCλ1 (but not pTCλ2) plasmid, the λ 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 λcro-plasmids depends on the interaction of the DNA-bound DnaA protein with the DNA-bound λ replication complex. The replication complex- DnaA-λ DNA structure may be directly related to the role of DnaA as the Cro- replacing negative regulator of λcro- plasmid replication.