Inheritance of the replication complex by one of two daughter copies during λ plasmid replication in Escherichia coli

Abstract

Direct measurement of DNA synthesis confirmed that λ plasmid replication proceeds for several hours in an amino acid-starved relA mutant of Escherichia coli, leading to plasmid amplification; this replication is λcro-independent, but requires the function of λO initiator in the absence of its synthesis. This suggests that after the assembly of the replication complex (RC) at oriλ the λO protein remains in this structure and the affinity of λO to oriλ is alleviated in the assembled RC allowing its movement along the DNA. During amino acid starvation the λ plasmid DNA synthesis per bacterial mass occurs at a constant level, as would be expected if the number of functioning RCs remained constant. This favors the idea that under these conditions the next replication round operates due to the activity of the RC inherited from the preceding round. Density shift experiments reveal indeed that, from two daughter plasmid copies synthesized after the onset of amino acid starvation only one is able to enter into the next round of replication. We infer that this is the plasmid copy that inherits the λO-enclosing RC from the previous replication round. Moreover, the same results of density shift experiments were obtained for plasmids synthesized before the onset of amino acid starvation, Therefore, we presume that in λ plasmid-harboring bacteria growing in nutrient medium, every second plasmid circle bears an RC that originates from the preceding round of replication. This structure has to be assembled de novo only on the daughter plasmid copy that does not inherit the parental RC. In the absence of λO initiator synthesis in amino acid-starved relA cells this process cannot occur, leaving as the only replication pathway that driven by the parental RC. Our results are discussed in relation to the model of regulation of λ plasmid replication.