A Supercos-1 library carrying chromosomal DNA of a plasmid-free derivative of Streptomyces coelicolor A3(2) was organized into an ordered encyclopaedia of overlapping clones by hybridization. The minimum set of overlapping clones representing the entire chromosome (with three short gaps) consists of 319 cosmids. The average insert size is 37.5 kb and the set of clones therefore divides the chromosome into 637 alternating unique and overlapping segments which have an average length of approx. 12.5 kb. More than 170 genes, gene clusters and other genetic markers were mapped to their specific segment by hybridization to the encyclopaedia. Genes could be cloned by direct transformation and complementation of S. coelicolor mutants with cosmids isolated from Escherichia coli, selecting for insertion into the chromosome by homologous recombination. As in other streptomycetes, the ends of the chromosome have long terminal inverted repeats.

SummaryThe complete nucleotide sequence of the large linear plasmid pSLA2-L in Streptomyces rochei strain 7434AN4 has been determined. pSLA2-L was found to be 210 614 bp long with a GC content of 72.8% and carries 143 open reading frames. It is especially noteworthy that three-quarters of the pSLA2-L DNA is occupied by secondary metabolism-related genes, namely two type I polyketide synthase (PKS) gene clusters for lankacidin and lankamycin, a mithramycin synthase-like type II PKS gene cluster, a carotenoid biosynthetic gene cluster and many regulatory genes. In particular, the lankacidin PKS is unique, because it may be a mixture of modular-and iterative-type PKSs and carries a fusion protein of non-ribosomal peptide synthetase and PKS. It is also interesting that all the homologues of the afsA , arpA , adpA and strR genes in the A-factor regulatory cascade in Streptomyces griseus were found on pSLA2-L, and disruption of the afsA homologue caused non-production of both lankacidin and lankamycin. These results, together with the finding of three possible replication origins at 50-63 kb from the right end, suggest that the present form of pSLA2-L might have been generated by a series of insertions of the biosynthetic gene clusters into the left side of the original plasmid.

A number of examples of circular plasmids with specific functions are known in both prokaryotes and eukaryotes. Several linear plasmids have also been identified, but these are all relatively small: large linear plasmids cannot be separated from chromosomal DNA by conventional techniques. There are several cases where the genetic evidence suggests that a character is encoded by a plasmid but no plasmid can be physically detected. This has been the case for antibiotic synthesis genes in Streptomyces; in particular a plasmid SCP1 in Streptomyces coelicolor has been shown to be involved in methylenomycin production by genetic evidence. We report here the application of orthogonal-field-alternation gel electrophoresis to the isolation of linear plasmids from Streptomyces. We have discovered a large linear plasmid of around 520 kilobases in Streptomyces lasaliensis and subsequently similar giant linear plasmids in other Streptomyces strains. We have confirmed that genes for methylenomycin biosynthesis are located on a series of giant linear plasmids in S. coelicolor. These observations may bear on the genetic variability and unstable genetic character of Streptomyces species.

SummaryThe sequencing of the entire genetic complement of Streptomyces coelicolor A3(2) has been completed with the determination of the 365 023 bp sequence of the linear plasmid SCP1. Remarkably, the functional distribution of SCP1 genes somewhat resembles that of the chromosome: predicted gene products/functions include ECF sigma factors, antibiotic biosynthesis, a gamma-butyrolactone signalling system, members of the actinomycete-specific Wbl class of regulatory proteins and 14 secreted proteins. Some of these genes are among the 18 that contain a TTA codon, making them targets for the developmentally important tRNA encoded by the bldA gene. RNA analysis and gene fusions showed that one of the TTAcontaining genes is part of a large bldA -dependent operon, the gene products of which include three proteins isolated from the spore surface by detergent washing (SapC, D and E), and several probable metabolic enzymes. SCP1 shows much evidence of recombinational interactions with other replicons and transposable elements during its history. For example, it has two sets of partitioning genes (which may explain why an integrated copy of SCP1 partially suppressed the defective partitioning of a parAB-deleted chromosome during sporulation). SCP1 carries a cluster of probable transfer determinants and genes encoding likely DNA polymerase III subunits, but it lacks an obvious candidate gene for the terminal protein associated with its ends. This may be related to atypical features of its end sequences.

Streptomyces rochei 7434AN4, a producer of lankacidin and lankamycin contains three large linear plasmids, pSLA2-L(200kb), M (100kb), and S (17 kb). Studies on the mutants of 7434AN4 having a different plasmid profile showed a parallel relationship between the presence of pSLA2-L and the production of both lankacidin and lankamycin. WhenpSLA2-Lwas transferred by protoplast fusion to S. rochei 2-39, a non-antibiotic-producing mutant of7434AN4which contained no detectable plasmid, the fusants gained the capacity to produce both antibiotics. From the physical maps of pSLA2-L and pSLA2-Ll , a deletion plasmid (160 kb) of pSLA2-L, the latter plasmid was determined to contain a symmetrical linear repeat composedof the right 80-kb part of pSLA2-L.Four other lankacidin-producing Streptomyces strains were also found to have distinctive large linear plasmids which hybridized with the pSLA2-L probe. These results support the involvement of pSLA2-L in the production of lankacidin and lankamycin in S. rochei 7434AN4. The plasmid SCP1, which carries the methylenomycin biosynthetic gene cluster in Streptomyces coelicolor A3 (2), was shown to be a giant linear plasmid of 350 kb by pulsed field gel electrophoresis (PFGE)12). In an effort to investigate the involvement of linear plasmids in antibiotic biosynthesis following SCP1, large linear plasmids were detected in five Streptomyces strains3). Amongthem, Streptomyces rochei 7434AN4had three linear plasmids of 200, 100 and 17 kb. This strain produces two structurally unrelated antibiotics, Iankacidin4'5), a 1 7-membered macrolide antibiotic and lankamycin6), a 1 6-membered macrolide antibiotic. The same strain was reported to possess plasmid pSLA2, which was the first linear plasmid isolated from bacteria7~9). The smallest of the threes plasmids was found to be identical to pSLA2. We named the three linear plasmids pSLA2-S, M, and L, respectively. Curing experiments of pSLA2-S by Hayakawa et al.7) suggested that pSLA2-S was involved in the lankacidin production.Based on these data, we selected S. rochei 7434AN4and other lankacidin-producing strains, to study the involvement of plasmid in antibiotic production. In this paper, we report the detection of large linear plasmids in all the lankacidin-producers tested and their homology by Southern hybridization. In particular, we describe the characterization of the three linear plasmids from S. rochei 7434AN4and the involvement of the largest plasmid, pSLA2-L, in the production of lankacidin and lankamycin.