Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.     

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.     

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.     

Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts.

A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transformation frequencies for both species were also influenced by the culture age before protoplast formation, the source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates. Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae, suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at frequencies of 10(6) to 10(7) transformants per micrograms of plasmid DNA.     

Inhibition of restriction in Streptomyces clavuligerus by heat treatment

Inefficient transformation of Streptomyces clavuligerus protoplasts by DNA from the plasmid pIJ702, isolated from S. lividans, was attributed to restriction in view of the observation that efficient transformation was observed using modified pIJ702 (isolated from S. clavuligerus). The restriction system could be partially inhibited by treating protoplasts at 45 degrees C prior to transformation. This treatment increased the transformation frequencies of pIJ702 DNA by 100-fold and was used to introduce other plasmids into S. clavuligerus.     

Protoplast transformation of glutamate-producing bacteria with plasmid DNA

A method for polyethylene glycol-induced protoplast transformation of glutamate-producing bacteria with plasmid DNA was established. Protoplasts were prepared from cells grown in the presence of penicillin by treatment with lysozyme in a hypertonic medium. The concentration of penicillin during growth affected the efficiency of formation, regeneration, and polyethylene glycol-induced DNA uptake of protoplasts. Regeneration of protoplasts was accomplished on a hypertonic agar medium containing sodium succinate and yeast extract. The spectinomycin and streptomycin resistance plasmid pCG4, originally from Corynebacterium glutamicum T250, could transform various glutamate-producing bacteria such as C. glutamicum, Corynebacterium herculis, Brevibacterium flavum, and Microbacterium ammoniaphilum. The plasmid was structurally unchanged and stably maintained in new hosts. The transformation frequency of most competent protoplasts with pCG4 DNA isolated from primary transformants was high (ca. 10(6) transformants per microgram of covalently closed circular DNA) but was still two orders of magnitude below the frequency of transfection with modified DNA of the bacteriophage phi CGI. The difference was ascribed to the involvement of regeneration in transformation.     

Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector.

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.     

Transformation ofStreptomyces avermitilis by plasmid DNA

Summary Polyethylene glycol (PEG) efficiently mediated the transformation ofStreptomyces avermitilis protoplasts by plasmid DNA to yield 10⁷ transformants per g of plasmid DNA. Under conditios in which the maximum transformation frequency was observed, the cotransformation frequency exceeded 10%. The number of transformants increased linearly with the amount of DNA and number ofS. avermitilis protoplasts. Relaxed and supercoiled, but not linear DNA transformed protoplasts efficiently. Dimethyl sulfoxide (DMSO)-mediated transformation of protoplasts was 1000-fold less efficient. PEG and, less efficiently, DMSO also mediated the transformation of whole cells ofS. avermitilis by DNA.     

High-frequency transformation of Brevibacterium lactofermentum protoplasts by plasmid DNA.

An efficient polyethylene glycol-assisted method for transformation of Brevibacterium lactofermentum protoplasts that uses plasmid vectors has been developed. Two small plasmids, pUL330 (5.2 kilobases) and pUL340 (5.8 kilobases), both containing the kanamycin resistance gene from transposon Tn5 and the replication origin of the natural plasmid pBL1 of B. lactofermentum, were selected as vectors. Supercoiled forms of the plasmids yielded a 100-fold higher transformation frequency than did linear forms. The optimal transformation frequency was achieved with 10 ng of DNA in 1 ml of transformation buffer. Higher concentrations of plasmid DNA resulted in a decrease in transformation frequency per microgram of DNA. Optimal transformation was obtained with 25 to 35% polyethylene glycol 6000. Under optimal conditions, 10(6) transformants per microgram of DNA were obtained.     

Plasmid transformation of Streptomyces tendae after heat attenuation of restriction

Streptomyces tendae ATCC 31160 produces nikkomycin, a fungicide and insecticide that inhibits chitin synthases. Exposure of S. tendae protoplasts to 50 degrees C for 30 min is required for transformation (10(2) thiostrepton-resistant transformants micrograms of DNA-1) with plasmid pIJ702 or pIJ680 from Streptomyces lividans. pIJ702 and pIJ680 DNA isolated from the S. tendae transformants is efficient (10(6) to 10(7) transformants micrograms of DNA-1) in subsequent transformations of S. tendae protoplasts generated at 30 degrees C. PstI fails to cut the single PstI site in pIJ702 and cuts only one of the two PstI sites in pIJ680 DNA isolated from S. tendae transformants. Digests of plasmid DNA mixtures showed that plasmid DNA from S. tendae does not inhibit PstI activity. pIJ702 and pIJ680 DNA from S. tendae transformants was used to transform S. lividans to show that plasmid DNA remains unchanged, except for modification at some PstI sites in S. tendae, as a consequence of passage through S. tendae. The DNA modification is lost when S. lividans is transformed with plasmid DNA from S. tendae transformants. Since S. tendae modifies only some PstI sites, it appears the modification (presumably restriction activity also) activity in S. tendae recognizes a sequence that includes or overlaps the PstI hexanucleotide recognition sequence.     

Improvement of transformation and electroduction in avermectin high-producer,<Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

Factors affecting the PEG-mediated transformation and electrotransformation of Streptomyces avermitilis protoplasts, an industrial avermectin high-producer, were evaluated. The maximum protoplast transformation efficiency under optimum conditions with PEG was 3 x 106 transformants per microg plasmid pIJ702 DNA. The efficiency of electrotransformation with the same plasmid the intact cells grown in medium with 0.5 mmol/L CaCl2, suspended in buffer with 0.5 mol/L sucrose +1 mmol/L MgCl2, and pulsed at an electric field strength of 10 kV/cm, 800 ohms, 25 microF, was of 2 x 10(3) transformants per microg DNA. When the cells were electroporated after mild lysozyme-treatment, the efficiency was up to 10(4) transformants per microg DNA. Electroporation of protoplasts and germlings had a lower efficiency (10(2) transformants per microg DNA). We report that electroporation under optimum conditions can be used for direct transfer of nonconjugative plasmid pIJ699 between two different Streptomyces species, S. avermitilis and S. lividans.     

High-efficiency transformation of Streptococcus lactis protoplasts by plasmid DNA.

Streptococcus lactis IL1403 protoplasts were transformed by plasmid pIL204 (5.5 kilobases), which conferred erythromycin resistance with an average efficiency of 5 X 10(6) transformants per microgram of supercoiled DNA. The procedure used and transformation efficiencies obtained were close to those described for Bacillus subtilis (G. Chang and S. N. Cohen, Mol. Gen. Genet. 168:111-115, 1979).     

High-efficiency transformation of Streptococcus lactis protoplasts by plasmid DNA

Streptococcus lactis IL1403 protoplasts were transformed by plasmid pIL204 (5.5 kilobases), which conferred erythromycin resistance with an average efficiency of 5 X 10(6) transformants per microgram of supercoiled DNA. The procedure used and transformation efficiencies obtained were close to those described for Bacillus subtilis (G. Chang and S. N. Cohen, Mol. Gen. Genet. 168:111-115, 1979).     

Optimal conditions for genetic transformation of the cyanobacterium Anacystis nidulans R2

Under optimal conditions, the cyanobacterium Anacystis nidulans R2 was transformed to ampicillin resistance at frequencies of greater than 10(7) transformants per microgram of plasmid (pCH1) donor DNA. No stringent period of competency was detected, and high frequencies of transformation were achieved with cultures at various growth stages. Transformation increased with time after addition of donor DNA up to 15 to 18 h. The peak of transformation efficiency (transformants/donor molecule) occurred at plasmid concentrations of 125 to 325 ng/ml with an ampicillin resistance donor plasmid (pCH1) and 300 to 625 ng/ml for chloramphenicol resistance conferred by plasmid pSG111. The efficiency of transformation was enhanced by excluding light during the incubation or by blocking photosynthesis with the electron transport inhibitor 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) or the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. Preincubation of cells in darkness for 15 to 18 h before addition of donor DNA significantly decreased transformation efficiency. Growth of cells in iron-deficient medium before transformation enhanced efficiency fourfold. These results were obtained with selection for ampicillin (pCH1 donor plasmid)- or chloramphenicol (pSG111 donor plasmid)-resistant transformants. Approximately 1,000 transformants per microgram were obtained when chromosomal DNA from an herbicide (DCMU)-resistant mutant was used as donor DNA. DCMU resistance was also transferred to recipient cells by using restriction fragments of chromosomal DNA from DCMU-resistant mutants. This procedure allowed size classes of fragments to be assayed for the presence of the DCMU resistance gene.     

Plasmid transformation of Streptomyces tendae after heat attenuation of restriction.

Streptomyces tendae ATCC 31160 produces nikkomycin, a fungicide and insecticide that inhibits chitin synthases. Exposure of S. tendae protoplasts to 50 degrees C for 30 min is required for transformation (10(2) thiostrepton-resistant transformants micrograms of DNA-1) with plasmid pIJ702 or pIJ680 from Streptomyces lividans. pIJ702 and pIJ680 DNA isolated from the S. tendae transformants is efficient (10(6) to 10(7) transformants micrograms of DNA-1) in subsequent transformations of S. tendae protoplasts generated at 30 degrees C. PstI fails to cut the single PstI site in pIJ702 and cuts only one of the two PstI sites in pIJ680 DNA isolated from S. tendae transformants. Digests of plasmid DNA mixtures showed that plasmid DNA from S. tendae does not inhibit PstI activity. pIJ702 and pIJ680 DNA from S. tendae transformants was used to transform S. lividans to show that plasmid DNA remains unchanged, except for modification at some PstI sites in S. tendae, as a consequence of passage through S. tendae. The DNA modification is lost when S. lividans is transformed with plasmid DNA from S. tendae transformants. Since S. tendae modifies only some PstI sites, it appears the modification (presumably restriction activity also) activity in S. tendae recognizes a sequence that includes or overlaps the PstI hexanucleotide recognition sequence.     

Transformation ofStreptomyces lincolnensis protoplasts with plasmid vectors

A method for the preparation and regeneration of protoplasts of Streptomyces lincolnensis is described. Mycelium in the early exponential phase appeared to be most suitable for this purpose and yielded up to 25% regenerated intact cells. Transformation of S. lincolnensis protoplasts was achieved using broad-host-range streptomycete plasmid vectors pIJ622, pMP66, pRS410 and pIJ943 constructed from replicons pIJ101, pSLG33 and SCP2. The efficiency of transformation was 3.10(3) transformants per micrograms plasmid DNA when (2-5).10(7) recipient protoplasts were used. Interspecific transformations showed that there is no efficient restriction system in S. lincolnensis that would limit the transfer of genetic information from S. lividans or E. coli.     

Transformation and transfection of anthracycline-producing streptomycetes.

Streptomyces peucetius and Streptomyces strain C5, producers or anthracycline antibiotics, were converted to protoplasts from vegetatively growing mycelia. Conditions are described for maximal protoplast formation (greater than 99%) and for regeneration frequencies of up to 13%. Streptomycete plasmids pIJ61, pIJ702, and pIJ922, from the replicons SLP1, pIJ101, and SCP2, respectively, were isolated from Streptomyces lividans 66 and successfully introduced into S. peucetius and Streptomyces strain C5 by polyethylene glycol-mediated protoplast transformation. Frequencies of up to 10(6) transformations X microgram of plasmid DNA-1 were achieved by these procedures. Analyses showed that the two anthracycline-producing strains can stably harbor the plasmids without deletion of plasmid sequences or loss of the plasmids for several transfers through selective media. Fragments of DNA from S. peucetius ligated into pIJ702 and introduced into Streptomyces strain C5 were stable after several transfers through selective media. Both anthracycline producers also were sensitive to infection and transfection by actinophages KC401 and KC515, clear plaque derivatives of bacteriophage phi C31. Optimal conditions were determined for the transfection of S. peucetius and Streptomyces strain C5 protoplasts with phi C31 KC401 and KC515 DNA with liposome-assisted, polyethylene glycol-mediated protoplast transfection.     

Transformation and transfection of anthracycline-producing streptomycetes

Streptomyces peucetius and Streptomyces strain C5, producers or anthracycline antibiotics, were converted to protoplasts from vegetatively growing mycelia. Conditions are described for maximal protoplast formation (greater than 99%) and for regeneration frequencies of up to 13%. Streptomycete plasmids pIJ61, pIJ702, and pIJ922, from the replicons SLP1, pIJ101, and SCP2, respectively, were isolated from Streptomyces lividans 66 and successfully introduced into S. peucetius and Streptomyces strain C5 by polyethylene glycol-mediated protoplast transformation. Frequencies of up to 10(6) transformations X microgram of plasmid DNA-1 were achieved by these procedures. Analyses showed that the two anthracycline-producing strains can stably harbor the plasmids without deletion of plasmid sequences or loss of the plasmids for several transfers through selective media. Fragments of DNA from S. peucetius ligated into pIJ702 and introduced into Streptomyces strain C5 were stable after several transfers through selective media. Both anthracycline producers also were sensitive to infection and transfection by actinophages KC401 and KC515, clear plaque derivatives of bacteriophage phi C31. Optimal conditions were determined for the transfection of S. peucetius and Streptomyces strain C5 protoplasts with phi C31 KC401 and KC515 DNA with liposome-assisted, polyethylene glycol-mediated protoplast transfection.     

Optimum conditions for efficient transformation ofStreptomyces venezuelae protoplasts

Summary Optimum conditions for protoplast regeneration and transformation ofStreptomyces venezuelae ETH 14630 have been established. Protoplasts from mycelium grown to the stationary phase and treated with lysozyme in P medium under mild conditions gave the best regeneration frequency. Transformation of protoplasts with naked DNA was very efficient using either polyethylene glycol of mol. wt. 4000 or 6000, at concentrations of 28.5% or 36% (w/v) respectively. About 10⁵ transformants/g DNA could be isolated using protoplasts derived from cells cultivated to the early exponential growth phase in LB medium containing 0.2%-0.6% glycine and subsequently treated at 30°–32°C with 20 mg lysozyme/ml in P medium for 30 min. Selection of the transformants occurred on MRYE plates containing less than 10⁵ regenerating protoplasts per plate. Higher protoplast densities considerably decreased the regeneration frequency of the transformants.     

Transformation of Penicillium chrysogenum with selection for increased resistance to benomyl

Incubation of protoplasts of Penicillium chrysogenum with the plasmid pBT-3 permitted selection of transformants on the basis of increased resistance to the anti-fungal agent benomyl. Transformants were obtained at a frequency of 1-2 per microgram of DNA. Southern analysis revealed that transformation had occurred by integration of vector sequences into the host genome.