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We report here the isolation and recombinational cloning of a large plasmid, pZL12, from endophytic Streptomyces sp. 9R-2. pZL12 comprises 90,435 bp, encoding 112 genes, 30 of which are organized in a large operon resembling bacteriophage genes. A replication locus (repA) and a conjugal transfer locus (traA-traC) were identified in pZL12. Surprisingly, the supernatant of a 9R-2 liquid culture containing partially purified phage particles infected 9R-2 cured of pZL12 (9R-2X) to form plaques, and a phage particle ([phi]ZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of [phi]ZLI2 virions were encoded by pZLI2 genes. Like bacteriophage P1, linear [phi]ZL12 DNA contained ends from a largely random pZLI2 sequence. There was also a hot end sequence in linear [PHI]ZL12. [PHI]ZL12 virions efficiently infected only one host, 9R-2X, but failed to infect and form plaques in 18 other Streptomyces strains. Some 9R-2X spores rescued from lysis by infection of [phi]ZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lyric phage [phi]ZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces. doi: 10.1128/JB.00123-10