A single-strand initiation (ssi) signal was detected on the Lactococcus lactis plasmid pGKV21 containing the replicon of pWV01 by its ability to complement the poor growth of an M13 phage derivative (M13⌬lac182) lacking the complementary-strand origin in Escherichia coli. This ssi signal was situated at the 229-nucleotide (nt) DdeI-DraI fragment and located within the 109 nt upstream of the nick site of the putative plus origin. SSI activity is orientation specific with respect to the direction of replication. We constructed an ssi signal-deleted plasmid and then examined the effects of the ssi signal on the conversion of the single-stranded replication intermediate to double-stranded plasmid DNA in E. coli. The plasmid lacking an ssi signal accumulated much more plasmid single-stranded DNA than the wild-type plasmid did. Moreover, deletion of this region caused a great reduction in plasmid copy number or plasmid maintenance. These results suggest that in E. coli, this ssi signal directs its lagging-strand synthesis as a minus origin of plasmid pGKV21. Primer RNA synthesis in vitro suggests that E. coli RNA polymerase directly recognizes the 229-nt ssi signal and synthesizes primer RNA dependent on the presence of E. coli single-stranded DNA binding (SSB) protein. This region contains two stem-loop structures, stem-loop I and stem-loop II. Deletion of stem-loop I portion results in loss of priming activity by E. coli RNA polymerase, suggesting that stem-loop I portion is essential for priming by E. coli RNA polymerase on the SSB-coated single-stranded DNA template.Primer RNA synthesis for the replication of Escherichia coli single-stranded DNA (ssDNA) phages may be accomplished by one of the three different modes of priming (19). Initiation mechanisms of complementary-strand synthesis have been distinguished on the basis of the minus-origin sequences and host function requirements. In each case, an RNA primer is synthesized prior to the initiation of DNA synthesis. This RNA primer is synthesized by primase and RNA polymerase in case of G4 and M13 phages, respectively (4, 7). In X174, conversion of ssDNA to double-stranded DNA (dsDNA) requires the formation of a primosome, a multiprotein complex composed of at least seven host proteins (29). Primer RNAs are synthesized at multiple sites located throughout the X174 genome due to the mobile nature of this protein complex (1,26,28). It was found that many plasmids isolated from E. coli contained specific nucleotide sequences required for priming DNA synthesis on an ssDNA template. These sequences have been called the single-strand initiation (ssi) signals. These ssi signals were identified by their ability to complement the poor growth of an ssDNA phage in which a great part of minus origin had been deleted (17,27). The ssi signals of E. coli plasmids were also classified into four major groups with respect to their priming mode: (i) G4 type (3, 23, 24, 32), (ii) X174 type (2, 15, 23-25), (iii) ABC type (22), and (iv) plasmid-specific type (RSF1010) (12-14).Many rep...