3 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 replic ates via a single-stranded intermediate by the rolling circle replicat ion (RCR) mechanism. We identified an unidirectional single-strand ori gin (SSO) of pSN22, ssol, where the initiation of second-strand synthe sis takes place, located between the spdA and traR genes in a noncodin g region which is functional in its natural orientation. The nucleotid e sequence of ssol is similar over 170 bp to the SSOs of the Streptomy ces plasmids PIJ101 and pJV1. A previous report described that a 548-b p BglII-SmaI fragment has an SSO activity (ori2; Kataoka el al., Mel. Gen. Genet. 242, 130-136, 1994). To our surprise, we discovered that o n pSN22, the SSO in the BglII-SmaI fragment is in the wrong, inactive, orientation and thus cannot be involved in the conversion of the sing le-stranded pSN22 replication intermediate to the double-stranded form of the plasmid. We revealed that this BglII-SmaI fragment contains tw o 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 bacte ria. Deletion of these hexanucleotide sequences caused loss of SSO act ivities. Our result shows that two types of SSOs, Streptomyces type an d Staphylococcus-like type, can function in Streptomyces lividans. (C) 1997 Academic Press.