Liposome-mediated transformation of streptomycetes by chromosomal DNA

Makins, J. F.; Holt, G.

doi:10.1038/293671a0

Cited by 39 publications

(13 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, many of these protocols gave variable results in our hands, possibly because of varying degree of trace contamination in the chemicals used. Several reports have dealt with the usefulness of liposomes in the delivery of transfecting and transforming DNA into recipient cells (3)(4)(5)(6)(7). In this study, the factor affecting the liposome-assisted transformation of S. lactis were examined.…”

Section: Discussionmentioning

confidence: 99%

“…Direct gene cloning in lactic acid streptococci requires high transformation efficiencies. It has been reported that in transformation experiments with Streptomyces protoplasts [3], yeast protoplasts [4], and transfection of mammalian cells with SV-40 DNA [5], entrapment of the DNA in liposomes increased the delivery of nucleic acids into the host cell as compared to non-aided internationalization of naked DNA. Rodicio and Chater [6] showed that DNA-free liposomes stimulated polyethylene glycol (PEG)-mediated transfection of Streptomyces protoplasts and Geis [7] observed that transfecting DNA could be introduced with a higher efficiency into S. lactis ssp.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Vossen¹

1988

FEMS Microbiology Letters

View full text Add to dashboard Cite

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Vossen¹

1988

FEMS Microbiology Letters

View full text Add to dashboard Cite

“…Previous reports of transformation of Streptomyces spp. by chromosomal DNA preparations do not appear to have been followed up (20,27,29).…”

Section: Fig 2 Transformation Of S Coelicolor J1501 By a Cosmid Comentioning

confidence: 99%

Denaturation of circular or linear DNA facilitates targeted integrative transformation of Streptomyces coelicolor A3(2): possible relevance to other organisms

Chater

1997

J Bacteriol

107

View full text Add to dashboard Cite

Using Streptomyces coelicolor A3(2) protoplasts, the number of transformants obtained by homologous recombination of incoming double-stranded circular DNA with the recipient chromosome was greatly stimulated by simple denaturation of the donor DNA. This procedure was very effective with inserts over a ca. 100-fold size range, the largest tested being ca. 40-kb inserts in cosmids. These observations led to transformation experiments with linearized cloned DNA and randomly sheared genomic DNA. In both cases, DNA denaturation led to significant levels of transformation. Most of the transformants had resulted from the predicted homologous recombination events. A number of genetic manipulations will be made easier or possible by these procedures.Streptomyces coelicolor A3(2), genetically the most studied of streptomycetes, serves as a model for the many important antibiotic-producing species of its genus and closely related genera such as Saccharopolyspora (10). Its morphologically complex colonies, consisting of a much-branched substrate mycelium and a spore-bearing aerial mycelium, have also attracted analysis. Many genetic tools have been developed for the study of S. coelicolor A3(2), including an almost complete ordered cosmid library of chromosomal fragments (32). However, because it typically takes at least 4 days for streptomycete colonies to develop through to abundant sporulation, genetic experiments involving multiple rounds of growth can be protracted. This is often the case in gene disruption and gene replacement (procedures which will become increasingly important after future extensive genome sequencing).Often, these experiments involve the manipulation of S. coelicolor DNA in an Escherichia coli plasmid that has no Streptomyces origin of replication, followed by introduction of the resulting constructs into S. coelicolor by protoplast transformation (22). Successful transformation depends on recombination between the S. coelicolor insert and its homolog in the S. coelicolor genome. The number of transformants obtained when S. coelicolor protoplasts are transformed with such E. coli double-stranded plasmids containing S. coelicolor DNA is typically rather low (usually in the range of 1 to 100 per g of plasmid DNA). Because double crossover events of the kind desired for many gene replacements usually (but not always [4,21]) make up only a minority of transformation events (typically, most are single crossovers, giving rise to integration of the vector), further rounds of growth and replication are often needed to detect the infrequent segregants in which a second crossover has occurred. Increasing the frequency of primary transformants, so that double crossover events could more often be represented at this stage, would thus be a significant benefit.Hilleman et al. (15) found that single-stranded circular E. coli plasmid DNA containing 4 kb of Streptomyces viridochromogenes DNA was 10 to 100 times more efficiently rescued in S. viridochromogenes by homologous recombination (up to 625 transformants per ...

show abstract

“…LMT was first reported in Escherichia coli (Fraley et al, 1979) and then in several other bacteria, including Gram-negative bacteria (Kawata et al, 2004), Gram-positive bacteria (Makins and Holt, 1981;Yu et al, 2001), and archaea (Metcalf et al, 1997). However, this method is not very effective in bacteria with cell walls, which blocks the passage of large molecules or vesicles, and has to be combined with other techniques such as competent cells/ CaCl 2 , protoplast preparation, electroporation or PEG/ CaCl 2 -mediation (Chai et al, 2013).…”

Section: Discussionmentioning

confidence: 99%

Highly-efficient liposome-mediated transformation system for the basidiomycetous fungus <i>Flammulina velutipes</i>

Shi

Chen

et al. 2017

J. Gen. Appl. Microbiol.

View full text Add to dashboard Cite

Flammulina velutipes is a well-known edible mushroom cultivated all over the world. However, because of the low transformation frequency, the expensive instruments required, and the complicated, time-consuming procedures necessary, there is insufficient genetic research on F. velutipes. In this study, we report a liposome-mediated transformation (LMT) system for the genetic transformation of F. velutipes. Using the LMT system, we obtained 82 ± 4 stable F. velutipes transformants per 10 5 protoplasts, which is a clear increase in transformation frequency compared to the other methods used. We were able to detect the expression of an EGFP reporter gene in the F. velutipes transformants using fluorescence imaging assays. Furthermore, we used this method to transfer the laccase gene into F. velutipes and found that the transcriptional level and enzymatic activity increased in these transformants. Mitotic stability analysis showed that all of the selected transformants remained mitotically stable, even after five successive rounds of sub-culturing. These results demonstrate a new transgenic approach that will facilitate F. velutipes research.

show abstract

Liposome-mediated transformation of streptomycetes by chromosomal DNA

Cited by 39 publications

References 11 publications

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Liposome-enhanced transformation of streptococcus lactis and plasmid transfer by intergeneric protoplast fusion of streptococcus lactis and bacillus subtilis

Denaturation of circular or linear DNA facilitates targeted integrative transformation of Streptomyces coelicolor A3(2): possible relevance to other organisms

Highly-efficient liposome-mediated transformation system for the basidiomycetous fungus <i>Flammulina velutipes</i>

Contact Info

Product

Resources

About