Three single-strand origins located on both strands of the Streptomyces rolling circle plasmid pSN22

pSN22 is an 11-kbp, high-copy-number Streptomyces plasmid 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 the spdA and traR genes in a noncoding region which is functional in its natural orientation. The nucleotide sequence of sso1 is similar over 170 bp to the SSOs of the Streptomyces plasmids pIJ101 and pJV1. A previous report described that a 548-bp BglII-SmaI fragment has an SSO activity (ori2; Kataoka et al., Mol. Gen. Genet. 242, 130-136, 1994). To our surprise, we discovered that on pSN22, the SSO in the BglII-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 this BglII-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 from Staphylococcus and the other several Gram-positive bacteria. Deletion of these hexanucleotide sequences caused loss of SSO activities. Our result shows that two types of SSOs, Streptomyces type and Staphylococcus-like type, can function in Streptomyces lividans.