Three Single-Strand Origins Located on Both Strands of theStreptomycesRolling Circle Plasmid pSN22

Abstract

pSN22 is an 11-kbp, high-copy-numberStreptomycesplasmid which replicates via a single-stranded intermediate by the rolling circle replication (RCR) mechanism. We identified an unidirectional single-strand origin (SSO) of pSN22,sso1,where the initiation of second-strand synthesis takes place, located between thespdAandtraRgenes in a noncoding region which is functional in its natural orientation. The nucleotide sequence ofsso1is similar over 170 bp to the SSOs of theStreptomycesplasmids pIJ101 and pJV1. A previous report described that a 548-bpBglII–SmaI fragment has an SSO activity (ori2;Kataokaet al., Mol. Gen. Genet.242,130–136, 1994). To our surprise, we discovered that on pSN22, the SSO in theBglII–SmaI fragment is in the wrong, inactive, orientation and thus cannot be involved in the conversion of the single-stranded pSN22 replication intermediate to the double-stranded form of the plasmid. We revealed that thisBglII–SmaI fragment contains two SSO fragments. Secondary structure analysis of these two SSOs showed similarity to the consensus TAGCGT which is conserved in SSOs of RCR plasmids fromStaphylococcusand the other several Gram-positive bacteria. Deletion of these hexanucleotide sequences caused loss of SSO activities. Our result shows that two types of SSOs,Streptomycestype andStaphylococcus-like type, can function inStreptomyces lividans.