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The protein synthesis inhibitor anisomycin features a unique benzylpyrrolidine system and exhibits diverse biological and pharmacologic activities. Its biosynthetic origin has remained obscure for more than 60 y, however. Here we report the identification of the biosynthetic gene cluster (BGC) of anisomycin in Streptomyces hygrospinosus var. beijingensis by a bioactivity-guided high-throughput screening method. Using a combination of bioinformatic analysis, reverse genetics, chemical analysis, and in vitro biochemical assays, we have identified a core four-gene ensemble responsible for the synthesis of the pyrrolidine system in anisomycin: aniQ , encoding a aminotransferase that catalyzes an initial deamination and a later reamination steps; aniP , encoding a transketolase implicated to bring together an glycolysis intermediate with 4-hydroxyphenylpyruvic acid to form the anisomycin molecular backbone; aniO , encoding a glycosyltransferase that catalyzes a cryptic glycosylation crucial for downstream enzyme processing; and aniN , encoding a bifunctional dehydrogenase that mediates multistep pyrrolidine formation. The results reveal a BGC for pyrrolidine alkaloid biosynthesis that is distinct from known bacterial alkaloid pathways, and provide the signature sequences that will facilitate the discovery of BGCs encoding novel pyrrolidine alkaloids in bacterial genomes. The biosynthetic insights from this study further set the foundation for biosynthetic engineering of pyrrolidine antibiotics., Competing Interests: The authors declare no conflict of interest.

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.

Actinomycete species from many genera often harbor linear plasmids and some contain linear chromosomes. A self-ligation and PCR-sequencing method was developed for identifying three novel telomere sequences of linear plasmids of Streptomyces and Mycobacterium. This and four previously described methods for actinomycetes telomere cloning and sequencing are discussed., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Objective: Large plasmid pCQ4 was detected in Streptomyces sp. W75 from Artemisia annua L. We clonined, sequenced, analyzed and characterized pCQ4., Methods: Southern hybridization was used to determine restriction map of pCQ4. To clone the full-length of pCQ4, conjugation and recombinational cloning in a BAC vector were used., Results: The complete nucleotide sequence of pCQ4 consisted of 84833-bp, encoding 129 ORFs which 40 ORFs resembled these of bacterial phages. W75 culture could infect W75 cured of pCQ4 and formed plaques on plate. Phage particle (phiCQ4) was observed by transmission electron microscopy. LinearphiCQ4 DNA was detected on pulsed-field gel electrophoresis. Comparison to Streptomyces plasmid-phage pZL12, genes encoding major phage structural proteins resembled that of pCQ4., Conclusion: Streptomyces plasmid pCQ4 could be transformed into lytic phagephiCQ4, and the phage segment on pCQ4 might be a mobile unit.

The complete nucleotide sequence including the novel telomere sequence of Streptomyces linear plasmid pSHK1 consists of 187,263-bp, 158 genes, in which 51 genes resemble those of the linear plasmid SCP1 of Streptomyces coelicolor A3(2), and 20 genes encode transposases. Strikingly, the repetitive CRISPRs (clustered regularly interspaced short palindromic repeats) and cas (CRISPR-associated) genes were found, including a cluster of eight cas genes, in the order cas2B-cas1B-cas3B-cas5-cas4-cas2A-cas1A-cas3A, bracketed by a pair of divergent CRISPRs, and five other dispersed CRISPRs. The cas2B-cas1B-cas3B-cas5 or cas4-cas2A-cas1A genes were co-transcribed. Protein-protein interactions between Cas5 and Cas1A, 2A, 2B, 3B were detected by yeast two-hybrids, indicating a critical role of Cas5 for the formation of protein complexes. By polymerase chain reaction and Southern hybridization, 12 cas4 genes including three on linear plasmids were found among 75 newly isolated Streptomyces strains. The paired-CRISPRs and bracketed cas were also conserved in several other Streptomyces or actinomycete species. However, unlike other bacteria, the CRISPRs-cas in pSHK1 could not provide immunity against introduction of phage ΦC31 and plasmid containing the particular spacers in Streptomyces.

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 (phiZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of phiZL12 virions were encoded by pZL12 genes. Like bacteriophage P1, linear phiZL12 DNA contained ends from a largely random pZL12 sequence. There was also a hot end sequence in linear phiZL12. phiZL12 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 phiZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lytic phage phiZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces.

Unlabelled: Strain 36R-2-1B was isolated from Allium fistulosum and identified as Streptomyces spp., harboring an approximate 280 kb linear plasmid designated pYY8L., Objective: Cloning, sequencing and analysis of telomere and internal replication origin of pYY8L., Methods: pYY8L telomere was cloned by a modified procedure--"alkaline treatment and enzyme digestion in gel". The internal replication origin of pYY8L was cloned by construction of a cosmid library and then subcloning., Results: An approximate 280 kb DNA band (pYY8L) of strain 36R-2-1B was detected by pulsed-field gel electrophoresis. The 152-bp telomere, containing six small palindromes and potentially forming complicated secondary structure, was cloned and sequenced. The replication origin of pYY8L was initially cloned on a cosmid and then sub-cloned as a 4891-bp autonomous replication sequence. This sequence was predicted to contain six genes, two of them resembling replication genes of Streptomyces linear plasmid SCP1, but their adjacent iterons were different., Conclusion: New telomere and internal replication origin of Streptomyces linear plasmid pYY8L were cloned and identified. This is the first example of report on telomere and replication genes of linear plasmid from endophytic Streptomyces.