Random inheritance of the replication complex by one of two daughter lambda plasmid copies after a replication round...

There are two pathways for replication of plasmids derived from bacteriophage lambda (so-called lambda plasmids) in Escherichia coli. One pathway is based on the assembly of the new replication complex at ori lambda, and the second requires activity of the replication complex inherited by one of two daughter plasmid copies after each replication round. Although these two replication pathways proceed at the same time in the host cell, we previously found conditions for specific elimination of the pathway based on the assembly of the new replication complex; thus, replication is restricted to that carried out by the heritable replication complex. These conditions are (i) the relaxed response to amino acid starvation and (ii) temperature upshift of the culture of cells harboring the lambda crotsPts1 plasmid. Here we asked whether the replication complex is inherited randomly by one of two daughter plasmid copies or whether the inheritance is preferred by one particular copy, that containing the parental DNA r strand or that bearing the l strand. We performed density shift experiments which allowed us to separate plasmid DNA molecules replicated by the heritable replication complex from those devoid of the replication complex and therefore not able to replicate. Then, [3H]thymidine-labelled plasmid DNA strands were separated and hybridized to membrane-bound ssDNA containing a fragment of either the r or l strand of lambda DNA. We found roughly equal efficiency of hybridization to both r and l strands in all experimental systems used. Therefore, we conclude that the lambda replication complex is randomly inherited by one of two daughter plasmid copies rather than preferentially inherited by either the copy carrying the parental r strand or that containing the l strand.