Genetic Recombination in Streptomyces fradiae by Protoplast Fusion and Cell Regeneration

Baltz, Richard H.

doi:10.1099/00221287-107-1-93

Cited by 145 publications

(75 citation statements)

References 18 publications

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“…Merkel, Eli Lilly) to maintain plasmid DNA. The actinomycete strains were grown in TS broth and fragmented by ultrasonic vibration (Baltz, 1978). Protoplast regeneration was carried out on modified R2 medium (Baltz, 1978), modified R2M medium (Matsushima & Baltz, 1988), R2LE medium (Matsushima & Baltz,l989), R2T20 medium (Yamamoto e t al., 1986) or R6 medium (Illing e t a/., 1989).…”

Section: Methodsmentioning

confidence: 99%

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Transformation of Saccharopolyspora spinosa protoplasts with plasmid DNA modified in vitro to avoid host restriction

Matsushima

Baltz

1994

Microbiology

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Indiana poi is, I n diana 46285, USA Saccharopolyspora spinosa protoplasts were not transformable by several different streptomycete plasmids, and S. spinosa was not a host for plaque formation by the Saccharopolyspora bacteriophages 4SE6,4SE45, 4SE57, 4SE60, 4SE69 or HP10. Extracts of S. spinosa contained DNA-modifying activities that blocked cleavage of plasmid DNA by Nael and Sall, and partially blocked cleavage by Ncol. Plasmid pOJ434, a derivative of the 5. spinosa plasmid pSAS1, was modified in vitro by incubation with an extract of S. spinosa to circumvent restriction. Under optimal conditions, 5. spinosa protoplasts were transformed to apramycin resistance by modified pOJ434 at frequencies of about lo4 per pg of DNA. Transformants contained pOJ434 primarily integrated in the chromosome.

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Section: Methodsmentioning

confidence: 99%

“….pinosa was grown in TS broth (Baltz, 1978) to prepare cell-free extracts for DNA modification experiments. TS agar (Baltz c3( Stonesifer, 1985) or R2LE agar supplemented with different antibiotics was used to determine the levels of antibiotic resistance expressed by S. spinosa.…”

Section: Methodsmentioning

confidence: 99%

Transformation of Saccharopolyspora spinosa protoplasts with plasmid DNA modified in vitro to avoid host restriction

Matsushima

Baltz

1994

Microbiology

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“…Extraction of lo9 protoplasts prepared under standard conditions with ethyl acetate followed by thin-layer chromatography and bioautography revealed the presence of the antibiotic in these cells. Autoinhibition of regenerating protoplasts was described for Streptomyces fradiae (Baltz, 1978) and Streptomyces acrimycini (Hopwood et al, 1977), but was not significant in the case of S. chrysomallus since, during regeneration of the individual strains, confluent lawns were observed.…”

Section: Resultsmentioning

confidence: 99%

“…Although conventional matings still seem to be the most reliable basis for the construction of chromosomal linkage groups, protoplast fusion has also been reported to be suitable in streptomycetes (Hopwood & Wright, 1978;Hopwood et al, 1977;Baltz, 1980). Moreover, this technique may be particularly useful for the construction of complex genotypes.…”

Section: Introductionmentioning

confidence: 99%

Studies of Protoplast Fusion in Streptomyces chrysomallus

et al. 1983

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Conditions for the regeneration of cells from protoplasts of Streptomyces chrysomallus, a producer of the peptide antibiotic actinomycin, are described. Regeneration of fusion products was most efficient at 27-30 "C on regeneration R2 medium (Okanishi et al., 1974) containing 0.25 M-sucrose. The addition of phosphate (150-300 mg 1-l) to the medium and incubation at 23 "C proved to be optimal for the regeneration of individual strains. Highest recombination frequencies after protoplast fusion were obtained by fusing protoplasts in the presence of 45 % (w/v) polyethylene glycol 6000. With strains that produce no, or little antibiotic, protoplasts must be present in excess in fusion mixtures in order to overcome inhibition of regeneration by the antibiotic-producing partner.

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“…were originally reported by Okanishi and co-workers. 2 The method developed for streptomycetes was then applied with uneven success to Micromonospora spp. 3 and Brevibacillus spp., 4 but it soon became clear that the protocol used later was species-or even strain-specific.…”

Section: Introductionmentioning

confidence: 99%

Protoplast preparation and reversion to the normal filamentous growth in antibiotic-producing uncommon actinomycetes

et al. 2010

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Protoplast preparation, regeneration and fusion represent essential tools for those poorly studied biotechnologically valuable microorganisms inapplicable with the current molecular biology protocols. The protoplast production and regeneration method developed for Planobispora rosea and using the combination of hen egg-white lysozyme (HEWL) and Streptomyces globisporus mutanolysin was applied to a set of antibiotic-producing filamentous actinomycetes belonging to the Streptosporangiaceae, Micromonosporaceae and Streptomycetaceae. 10 7 -10 9 protoplasts were obtained from 100 ml of culture, after incubation times in the digestion solution ranging from a few hours to 1 or 2 days depending on the strain. The efficiency of protoplast reversion to the normal filamentous growth varied from 0.1 to nearly 50%. Analysis of cell wall peptidoglycan in three representative strains (Nonomuraea sp. ATCC 39727, Actinoplanes teichomyceticus ATCC 31121 and Streptomyces coelicolor A3(2)) has evidenced structural variations in the glycan strand and in the peptide chain, which may account for the different response to cell digestion and protoplast regeneration treatments. Keywords: antibiotics; bacterial cell wall; glycopeptides; protoplast INTRODUCTIONThe so-called rare or uncommon actinomycetes include a group of filamentous actinomycetes other than Streptomyces spp., which are quite difficult to isolate, cultivate and genetically manipulate. 1 Members of these poorly represented and difficult-to-handle group of microorganisms produce valuable antibiotics. However, the study and the following cost-effective exploitation of uncommon actinomycetes have been slow because of the lack of genetic tools, which hamper strain and product improvement. As mobile genetic elements and conjugation systems are poorly characterized in rare actinomycetes, a crucial methodology to achieve their transformation by exogenous DNA or to recombine whole genomes arising from different cell lines (Whole Genome Shuffling-WGS) is based on protoplast manipulation and fusion. Protoplast preparation and regeneration in Streptomyces spp. were originally reported by Okanishi and co-workers. 2 The method developed for streptomycetes was then applied with uneven success to Micromonospora spp. 3 and Brevibacillus spp., 4 but it soon became clear that the protocol used later was species-or even strain-specific. In other industrially valuable actinomycetes, ad hoc techniques have been developed in some cases, 5-8 but methods endowed with a broad applicability and robustness to be applied for DNA recombination and WGS are not available. A possible explanation for the different response to protocols of protoplast

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Genetic Recombination in Streptomyces fradiae by Protoplast Fusion and Cell Regeneration

Cited by 145 publications

References 18 publications

Transformation of Saccharopolyspora spinosa protoplasts with plasmid DNA modified in vitro to avoid host restriction

Transformation of Saccharopolyspora spinosa protoplasts with plasmid DNA modified in vitro to avoid host restriction

Studies of Protoplast Fusion in Streptomyces chrysomallus

Protoplast preparation and reversion to the normal filamentous growth in antibiotic-producing uncommon actinomycetes

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