The minimal replicon of a Streptomycete plasmid produces an ultrahigh level of plasmid DNA

Larson, Jeffrey; Hershberger, Charles L.

doi:10.1016/0147-619x(86)90038-7

Cited by 123 publications

(83 citation statements)

References 30 publications

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“…2c) was subcloned in the unique SmaI site of pHJL400, an E. Polyketide synthase genes for jadomycin synthesis the partition function of the SCP2* parent (Larson & Hershberger, 1986). The recombinant plasmid (p JV62) isolated from E. coli TG1 transformed S. venexzlelae ISP5230 to thiostrepton resistance (the marker in pH JL400 used for selection) with very low efficiency.…”

Section: Gene Disruptionmentioning

confidence: 99%

Cloning and characterization of polyketide synthase genes for jadomycin B biosynthesis in Streptomyces venezuelae ISP5230

Han

Yang²,

Ramalingam³

et al. 1994

Microbiology

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Hybridizing fragments in the genomic DNA of Streptomyces venezuelae lSP5230, which produces the jadomycin group of angucycline antibiotics, were detected by probing with act/ DNA from Streptomyces coelicolor A3(2). The hybridizing regions were isolated from a 16.5 kb insert of S. venezuelae DNA recovered from a genomic library cloned in a A replacement vector. Subcloning and sequencing of a 4 8 kb segment of the insert, containing regions hybridizing to act/// as well as act/, identified five open reading frames (ORFs). The deduced polypeptide products of the ORFs closely resemble in sequence the components of streptomycete t y p e 4 polyketide synthases (PKSs) : the ORFI product corresponds to the ketoacyl synthase, and the ORF2 product to a polypeptide closely related to the ketoacyl synthase and involved in determining chain length; the ORF3 product matches the acyl carrier protein; ORF4 encodes a bifunctional cyclase/dehydrase; and ORF5 encodes a ketoreductase. Integration into the chromosomal DNA of a plasmid containing a segment of the ORF2-ORF4 region severely depressed jadomycin B biosynthesis; since the integrant showed no change in growth or spore pigmentation, the cloned PKS genes are presumed to encode enzymes in the pathway for jadomycin biosynthesis.

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Section: Gene Disruptionmentioning

confidence: 99%

Cloning and characterization of polyketide synthase genes for jadomycin B biosynthesis in Streptomyces venezuelae ISP5230

Han

Yang²,

Ramalingam³

et al. 1994

Microbiology

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“…The Streptomyes-E. coli bifunctional vector pH JL400 (Larson & Hershberger, 1986), which is segregationally unstable in S. venexuelae, was modified to obtain the replacement vectors pDQ402 and pDQ403 by inserting in both orientations a DNA fragment carrying bca disrupted at its Sall site with a gene conferring apramycin resistance (AprR). To construct the insert, pSF2201 was linearized by digestion with XbaI and PstI, and the single-strand Bromoperoxidase-catalase from S. veneatrelae overhangs were filled by incubation with the Klenow fragment and deoxynucleotide triphosphates.…”

Section: Methodsmentioning

confidence: 99%

“…Enriched genomic libraries were constructed in the high-copy-number plasmid vector pUC18 (Yanisch-Perron et al, 1985). Plasmids PI 5486 (Ward et al, 1986) and pH JL400 (Larson & Hershberger, 1986) were used in subcloning for heterologous expression and gene disruption. Transformants carrying pUCl8 and derivative plasmids were selected on agar media containing ampicillin (100 pg ml-'), 5-bromo-4-chloro-3-indolyl-/?-~-galactopyranoside (X-Gal; 40 pg ml-l) and 0.2 mM isopropyl-/?-D-thiogalactopyranoside (IPTG).…”

Section: Methodsmentioning

confidence: 99%

Cloning, sequencing and disruption of a bromoperoxidase-catalase gene in Streptomyces venezuelae: evidence that it is not required for chlorination in chloramphenicol biosynthesis

et al. 1996

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Genomic DNA libraries of Streptomyces venezuelae lSP5230 and of a mutant blocked at the chlorination step of chloramphenicol biosynthesis were probed by hybridization with a synthetic oligonucleotide corresponding t o the Nterminal amino acid sequence of a bromoperoxidase-catalase purified from the wild-type strain. Hybridizing fragments obtained from the two strains were cloned and sequenced. Analysis of the nucleotide sequences demonstrated that the fragments contained the same 1449 bp open reading frame with no differences in nucleotide sequence. The deduced polypeptide encoded 483 amino acids with a calculated M, of 54200; the N-terminal sequence was identical to that of the bromoperoxidase-catalase purified from wild-type S. venezuelae. Comparison of the amino acid sequence predicted for the cloned bromoperoxidase-catalase gene ( k a ) with database protein sequences showed a significant similarity to a group of prokaryotic and eukaryotic catalases, but none to other peroxidases or haloperoxidases. Replacement of the bca gene in the wild-type strain of S. venezuelae with a copy disrupted by insertion of a DNA fragment encoding apramycin resistance did not prevent chloramphenicol production. The results suggest that the role of the enzyme in S. venezuelae is related to its activity as a catalase rather than as a halogenating agent.

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“…A library of S. aki_yoshiensis DNA fragments in the Streptomyces-E. coli shuttle vector pH JL400 (Larson & Hershberger, 1986) was used to transform the E. coli asd mutant strain CGSC 6212. Screening approximately 39 000 transformants for prototrophy yielded one Asd' colony, from which we recovered a 17 kb plasmid (pJV21; Fig.…”

Section: Cloning Of Asd From 5 Akiyoshiensismentioning

confidence: 99%

“…Sized 6-15 kb fragments from Sau3AI partial digestion of S. akboshiensis genomic DNA were ligated behind the lac promoter in pHJL400 (Larson & Hershberger, 1986) linearized at its BamHI site and dephosphorylated with calfintestinal alkaline phosphatase (Promega DNA sequencing and sequence analysis. The DNA inserts in pJV24 and pJV36 were each subcloned in the phagemids pBluescript I1 SK( +) and SK( -), allowing both strands to be sequenced; nested deletions were generated by the DNase I method (Sambrook e t al., 1989).…”

Section: Introductionmentioning

confidence: 99%

Streptomyces akiyoshiensis differs from other Gram-positive bacteria in the organization of a core biosynthetic pathway gene for aspartate family amino acids

Vining

1996

Microbiology

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A partial Sau3Al digest of genomic DNA from Streptomyces akiyoshiensis was cloned in a Streptomyces-Escherichia coli shuttle vector, and the recombinant plasmids were used to transform E. coli CGSC 6212, which carries a mutation in the gene for aspartate semialdehyde dehydrogenase (Asd). One of 39000 transformants tested grew on LB medium lacking diaminopimelate. A 17 kb plasmid (pJV21) isolated from this strain conferred prototrophy when used to transform E. coli CGSC 6212. The gene responsible was located on a 2.2 kb DNA fragment by subcloning. Nucleotide sequencing and codon preference analysis of the subcloned insert and of the 3.3 kb insert in the Asd--complementing plasmid pJV36 located three complete and two incomplete open reading frames (ORFs). One of these (ORF3), encoding a polypeptide of 338 amino acids (M, 354848 was identified as the gene for Asd by comparing i t s sequence with database sequences of asd from other bacteria. The inability of pJV30, in which a segment of ORF3 had been deleted, to transform E. coli CGSC 6212 to prototrophy supported this assignment. Southern hybridization indicated that the sequenced region of the cloned DNA fragment represented a continuous segment of the 5. akiyoshiensis chromosome. The deduced amino acid sequences of the ORFs adjacent to asd showed no similarity to sequences for aspartate kinase (Ask); also, transformation with plasmids containing asd and adjacent regions from the 5. akiyoshiensis chromosome did not complement the ask mutant E. coli CGSC 5074. It is concluded that asd and ask in 5. akiyoshiensis are not present in an operon, and thus are organized differently from these genes in the Gram-positive bacteria previously examined.

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The minimal replicon of a Streptomycete plasmid produces an ultrahigh level of plasmid DNA

Cited by 123 publications

References 30 publications

Cloning and characterization of polyketide synthase genes for jadomycin B biosynthesis in Streptomyces venezuelae ISP5230

Cloning and characterization of polyketide synthase genes for jadomycin B biosynthesis in Streptomyces venezuelae ISP5230

Cloning, sequencing and disruption of a bromoperoxidase-catalase gene in Streptomyces venezuelae: evidence that it is not required for chlorination in chloramphenicol biosynthesis

Streptomyces akiyoshiensis differs from other Gram-positive bacteria in the organization of a core biosynthetic pathway gene for aspartate family amino acids

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