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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

Abstract Background Streptomyces species are widely distributed in natural habitats, such as soils, lakes, plants and some extreme environments. Replication loci of several Streptomyces theta-type plasmids have been reported, but are not characterized in details. Conjugation loci of some Streptomyces rolling-circle-type plasmids are identified and mechanism of conjugal transferring are described. Results We report the detection of a widely distributed Streptomyces strain Y27 and its indigenous plasmid pWTY27 from fourteen plants and four soil samples cross China by both culturing and nonculturing methods. The complete nucleotide sequence of pWTY27 consisted of 14,288 bp. A basic locus for plasmid replication comprised repAB genes and an adjacent iteron sequence, to a long inverted-repeat (ca. 105 bp) of which the RepA protein bound specifically in vitro, suggesting that RepA may recognize a second structure (e.g. a long stem-loop) of the iteron DNA. A plasmid containing the locus propagated in linear mode when the telomeres of a linear plasmid were attached, indicating a bi-directional replication mode for pWTY27. As for rolling-circle plasmids, a single traA gene and a clt sequence (covering 16 bp within traA and its adjacent 159 bp) on pWTY27 were required for plasmid transfer. TraA recognized and bound specifically to the two regions of the clt sequence, one containing all the four DC1 of 7 bp (TGACACC) and one DC2 (CCCGCCC) and most of IC1, and another covering two DC2 and part of IC1, suggesting formation of a high-ordered DNA-protein complex. Conclusions This work (i) isolates a widespread Streptomyces strain Y27 and sequences its indigenous theta-type plasmid pWTY27; (ii) identifies the replication and conjugation loci of pWTY27 and; (iii) characterizes the binding sequences of the RepA and TraA proteins.

The catechol 2,3-dioxygenase (C23O) gene in naphthalene catabolic plasmid pND6-1 of Pseudomonas sp. ND6 was cloned and sequenced. The C23O gene was consisted of 924 nucleotides and encoded a polypeptide of molecular weight 36 kDa containing 307 amino acid residues. The C23O of Pseudomonas sp. ND6 exhibited 93% and 89% identities in amino acid sequence with C23Os encoded by naphthalene catabolic plasmid NAH7 from Pseudomonas putida G7 and the chromosome of Pseudomonas stutzeri AN10 respectively. The Pseudomonas sp. ND6 C23O gene was overexpressed in Escherichia coli DH 5alpha using the lac promoter of pUC18, and its gene product was purified by DEAE-Sephacel and Phenyl-Sepharose CL-4B chromatography. The enzymology experiments indicated that the specific activity and thermostability of C23O from Pseudomonas sp. ND6 were better than those of C23O from Pseudomonas putida G7.