DETERMINATION OF THE SINGLE-STRAND ORIGIN OF SHIGELLA-SONNEI PLASMID PKYM

The Shigella sonnei plasmid pKYM replicates by a rolling-circle mechan ism in Escherichia coli. A 571 nucleotides HincII restriction fragment of the pKYM DNA harbors two potential hairpin loops (I and II). We cl oned the fragment into a -ori defective M13 vector phage, M13 Delta la c183. The chimera phage, MDKY5, showed a larger plaque size, and incre ased phage yield and rate of progeny replicative form DNA. (RF) synthe sis. Rifampicin reduced rate of conversion of the single- to double-st randed RF DNA. In addition, we introduced nucleotide deletions within the cloned pKYM DNA, by Bal31 nuclease digestion. Each of the deletion mutants thus constructed was lacking in a sequence containing the hai rpin loops and formed smaller plaques. The in vivo analyses revealed t hat a 136 nucleotides sequence containing the two hairpins I and II is the pKYM minus origin for complementary strand synthesis (single stra nd origin, referred to as SSO) and harbors a recognition site(s) by ho st E. coli RNA polymerase, for primer RNA synthesis. Moreover, we foun d a 24 nt sequence, upstream of the SSO domain having 83% homology to the recombination site A (RS(A)) which functions in plasmid sitespecif ic recombination and/or transfer.