Streptomyces lavendulae IMRU 3455 contains two large linear plasmids designated pSLV45 (45 kb) and pSLV195 (195 kb). A cosmid, pSPRX604, containing 42 kb from pSLV45 was cloned and sequenced. pSLV45 was tagged with a hygromycin-resistance marker by homologous recombination to generate the derivatives pSLV45.680 and pSLV45.681. An apramycin-resistance marker was introduced into S. lavendulae IMRU 467 using the pSPR910 integration vector to yield the recipient strain SPW910. The self-transmissible nature of pSLV45 was determined by transfer of pSLV45.680 and pSLV45.681 from the donor strains SPW680 and SPW681 into the recipient strain SPW910. Southern analysis indicated the presence of hygromycin-and pSLV45-hybridizing sequences within SPW910 exconjugants. PFGE analysis confirmed pSLV45.680 and pSLV45.681 were transferred intact and formed freely replicating linear plasmids. Sequence analysis of pSPRX604 revealed genes predicted to be involved in plasmid transfer, partitioning and regulation. The transfer of the linear plasmid pSLV45 from S. lavendulae IMRU 3455 into S. lavendulae IMRU 467 may allow the development of pSLV45 as an actinomycete-to-actinomycete conjugative shuttle vector.
INTRODUCTIONLinear plasmids with 59 terminally attached proteins (TPs) and terminal inverted repeats (TIRs) have been found in bacteria, plants, filamentous fungi and yeasts, and often within actinomycetes (Griffiths, 1995;Hinnebusch & Tilly, 1993;Meinhardt et al., 1990;Rohe et al., 1992). Actinomycete linear plasmids pSLA1 (17 kb) and pSLA2 (17 kb) were first detected in the lankacidin-producer Streptomyces sp. 7434-AN4 and in Streptomyces rochei, respectively (Hayakawa et al., 1979;Hirochika & Sakaguchi, 1982;Hirochika et al., 1984). Both plasmids were shown to contain TIRs and TPs (Hirochika & Sakaguchi, 1982;Hirochika et al., 1984). With the advent of PFGE analysis, other actinomycete linear plasmids have been identified including Streptomyces lividans SLP2 (50 kb), Streptomyces clavuligerus pSCL1 (12 kb), Streptomyces avermitilis pSA1 and pSA2 (100 kb and 250 kb), Streptomyces rimosus pZG101 (387 kb), Streptomyces lasaliensis pKSL (520 kb) and Streptomyces coelicolor SCP1 (363 kb) Evans et al., 1994;Gravius et al., 1994;Keen et al., 1988;Kinashi & Shimaji, 1987;Kinashi & Shimaji-Murayama, 1991;Kinashi et al., 1994). All these plasmids were found to contain TIRs and TPs. In addition, both pSLA2 and SLP2 were shown to contain an internal origin of replication for bi-directional replication of DNA (Chang & Cohen, 1994;Chang et al., 1996;Huang et al., 2003).Unlike the chromosomes of most bacteria, which are circular in nature, the chromosomes of actinomycetes are linear (Lezhava et al., 1995;Lin et al., 1993). This was first demonstrated in S. lividans and has now been established for several other actinomycetes including S. coelicolor, Streptomyces griseus and Saccharopolyspora erythraea (formerly Streptomyces erythraeus) (Lezhava et al., 1995;Lin et al., 1993;Reeves et al., 1998). All actinomycete chromosomes characterized to da...