Transformation of plasmid DNA into Streptomyces at high frequency

Bibb, Mervyn J.; Ward, Jacob; Hopwood, David A.

doi:10.1038/274398a0

Cited by 280 publications

(128 citation statements)

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“…Expression studies of the vdc genes in Streptomyces lividans 1326 were performed using pIJ680 This study (Hopwood et al, 1985), a vector that places target genes under the control of the aminoglycoside phosphotransferase (aph) constitutive promoter, and pIJ702 (Katz et al, 1983), which provides the weaker constitutive tyrosinase (mel) promoter. S. lividans 1326 was converted to protoplasts and transformed according to published methods (Bibb et al, 1978 ;Thompson et al, 1982). Protoplasts were plated on R5 solid medium (Thompson et al, 1980) and allowed to regenerate for 14 h before transformants were selected by an overlay of soft nutrient agar containing thiostrepton to achieve a final concentration of 50 µg thiostrepton ml − " per plate.…”

Section: Methodsmentioning

confidence: 99%

Characterization of a vanillic acid non-oxidative decarboxylation gene cluster from Streptomyces sp. D7 The GenBank accession number for the sequence reported in this paper is AF134589.

1999

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The genetics of non-oxidative decarboxylation of aromatic acids are poorly understood in both prokaryotes and eukaryotes. Although such reactions have been observed in numerous micro-organisms acting on a variety of substrates, the genes encoding enzymes responsible for these processes have not, to our knowledge, been reported in the literature. Here, the isolation of a streptomycete from soil (Streptomyces sp. D7) which efficiently converts 4-hydroxy-3-methoxybenzoic acid (vanillic acid) to 2-methoxyphenol (guaiacol) is described. Protein two-dimensional gel analysis revealed that several proteins were synthesized in response to vanillic acid. One of these was characterized by partial amino-terminal sequencing, leading to the cloning of a gene cluster from a genomic DNA lambda phage library, consisting of three ORFs, vdcB (602 bp), vdcC (1424 bp) and vdcD (239 bp). Protein sequence comparisons suggest that the product of vdcB (201 aa) is similar to phenylacrylate decarboxylase of yeast ; the putative products of vdcC (475 aa) and vdcD (80 aa) are similar to hypothetical proteins of unknown function from various micro-organisms, and are found in a similar cluster in Bacillus subtilis. Northern blot analysis revealed the synthesis of a 2 5 kb mRNA transcript in vanillic-acid-induced cells, suggesting that the cluster is under the control of a single inducible promoter. Expression of the entire vdc gene cluster in Streptomyces lividans 1326 as a heterologous host resulted in that strain acquiring the ability to decarboxylate vanillic acid to guaiacol non-oxidatively. Both Streptomyces sp. strain D7 and recombinant S. lividans 1326 expressing the vdc gene cluster do not, however, decarboxylate structurally similar aromatic acids, suggesting that the system is specific for vanillic acid. This catabolic system may be useful as a component for pathway engineering research focused towards the production of valuable chemicals from forestry and agricultural by-products.

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Section: Methodsmentioning

confidence: 99%

Characterization of a vanillic acid non-oxidative decarboxylation gene cluster from Streptomyces sp. D7 The GenBank accession number for the sequence reported in this paper is AF134589.

1999

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“…They were revealed by the appearance of "pocks," circular zones within an initially plasmid-free culture surrounding a coinoculated plasmid-containing spore ( Figure 5) and reflecting plasmid colonization of the recipient mycelium, assuming this led to growth retardation or perhaps, as originally proposed, even death of some hyphae (14,15). Smallpock mutations identified the spd genes, which were postulated to promote migration of plasmid copies inside the recipient mycelium (71), a hypothesis that has been universally adopted but not proven.…”

Section: Plasmid-mediated Dna Transfermentioning

confidence: 99%

Soil To Genomics: The Streptomyces Chromosome

2006

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The 8-9-Mb Streptomyces chromosome is linear, with a "core" containing essential genes and "arms" carrying conditionally adaptive genes that can sustain large deletions in the laboratory. Bidirectional chromosome replication from a central oriC is completed by "endpatching," primed from terminal proteins covalently bound to the free 5 -ends. Plasmid-mediated conjugation involves movement of double-stranded DNA by proteins resembling other bacterial motor proteins, probably via hyphal tip fusion, mediated by these transfer proteins. Circular plasmids probably transfer chromosomes by transient integration, but linear plasmids may lead the donor chromosome end-first into the recipient by noncovalent association of ends. Transfer of complete chromosomes may be the rule. The recipient mycelium is colonized by intramycelial spreading of plasmid copies, under the control of plasmid-borne "spread" genes. Chromosome partition into prespore compartments of the aerial mycelium is controlled in part by actin-and tubulin-like proteins, resembling MreB and FtsZ of other bacteria.

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“…The obtained recombinant plasmid was sequenced and designated as pUC702/plb. The transformation techniques for S. lividans followed the methods of Bibb et al [58] and Thompson et al [59]. Transformants were screened using lecithin-emulsified nutrient plates [53].…”

Section: Nucleotide and Peptide Sequence Accession Numbermentioning

confidence: 99%

A novel phospholipase B from Streptomyces sp. NA684 – purification, characterization, gene cloning, extracellular production and prediction of the catalytic residues

et al. 2013

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A novel metal ion-independent phospholipase B (PLB 684 ) from Streptomyces sp. strain NA684 was purified 264-fold from the culture supernatant with 2.85% recovery (6330 UÁmg protein À1 ). The enzyme functions as a monomer with a molecular mass of 38.9 kDa. Maximum activity was found at pH 8.4 and 50°C. The substrate specificity was in the order: phosphatidylcholine ≥ phosphatidic acid ≥ lysophosphatidylcholine > phosphatidylserine > phosphatidylinositol > phosphatidylglycerol. The enzyme did not hydrolyze phosphatidylethanolamine, tristearin and dipalmitin. PLB 684 hydrolyzed lysophosphatidylcholine and diacylphosphatidylcholine, and lysophosphatidylcholine was primarily produced during the early stages of phosphatidylcholine hydrolysis. The apparent K m , V max and k cat for hydrolysis of dimyristoyl phosphatidic acid were 14.5 mM, 15.8 mmolÁmin À1 Ámg protein À1 and 1.02 9 10 4 s À1 , respectively. The positional specificity of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine hydrolysis was investigated using GC. In the reaction equilibrium, the molar ratio of released fatty acids (sn-1 : sn-2) was 45 : 55. The ORF of the gene is 1239 bp in length and codes for a 30-amino acid signal peptide and a 382-amino acid mature enzyme. The deduced amino acid sequence of PLB 684 shows 60% identity to a uncharacterized protein of Streptomyces auratus AGR0001 (UniProt accession number: J1RQY0). The extracellular production of PLB 684 was achieved using a pUC702 expression vector and Streptomyces lividans as the host. Mutagenesis analysis showed that Ser12 is essential for the catalytic function of PLB 684 and that the active site may include residues Ser330 and His332.

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Transformation of plasmid DNA into Streptomyces at high frequency

Cited by 280 publications

References 13 publications

Characterization of a vanillic acid non-oxidative decarboxylation gene cluster from Streptomyces sp. D7 The GenBank accession number for the sequence reported in this paper is AF134589.

Characterization of a vanillic acid non-oxidative decarboxylation gene cluster from Streptomyces sp. D7 The GenBank accession number for the sequence reported in this paper is AF134589.

Soil To Genomics: The Streptomyces Chromosome

A novel phospholipase B from Streptomyces sp. NA684 – purification, characterization, gene cloning, extracellular production and prediction of the catalytic residues

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