Improvement of transformation and electroduction

Folia Microbiol. 49 (4), 399–405 (2004) http://www.biomed.cas.cz/mbu/folia/

Improvement of Transformation and Electroduction in Avermectin High-Producer, Streptomyces avermitilis

WEI GONG, WEIHONG JIANG, YUNLIU YANG*, JUISHEN CHIAO

Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200 032, China fax 86 21 5492 4015

e-mail ylyang@iris.ispp.ac.cn

Received 30 June 2003

Revised version 6 January 2004

ABSTRACT. Factors affecting the PEG-mediated transformation and electrotransformation of Streptomy-ces avermitilis protoplasts, an industrial avermectin high-producer, were evaluated. The maximum protoplast transformation efficiency under optimum conditions with PEG was 3 × 106 transformants per μg plasmid pIJ702 DNA. The efficiency of electrotransformation with the same plasmid the intact cells grown in me-dium 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 ȍ, 25 μF, was of 2 × 103 transformants per μg DNA. When the cells were electroporated after mild lysozyme-treatment, the efficiency was up to 104 transformants per μg DNA. Electroporation of protoplasts and germlings had a lower efficiency (102 transformants per μg 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.

Avermectins (Avm) produced by Streptomyces avermitilis are composed of 8 components of 16-mem-bered macrocyclic lactones. Because of its excellent anthelminthic and insecticidal activities, avermectin is widely used for treatment of diseases caused by nematodes and arthropods in veterinary and agricultural fields (Burg et al. 1979). The biosynthetic pathway of Avm was elucidated (Ikeda and Omura 1997) and the gene cluster for its biosynthesis was cloned and sequenced (Ikeda et al. 1999); this provided favorable conditions for further strain improvement. The development of methods for gene transfer may play an impor-tant role in the genetic manipulation of S. avermitilis. Since Bibb et al. (1978) reported a transformation procedure for Streptomyces coelicolor using protoplasts and polyethylene glycol (PEG), the PEG-mediated transformation of protoplasts has become a common technique applicable for introducing exogenous DNA into the cells of many Streptomyces spp. (Pigac and Schrempf 1982; Hopwood et al. 1985; Matsushima and Baltz 1985; MacNeil and Klapko 1987). However, this procedure has not provided the satisfactory transformation frequency in our avermectin high-production strain MMR630; we obtained less than 1000 transformants per μg of plasmid DNA. Moreover, Lee et al. (2000) and Hwang et al. (2001) indicated that the formation and regeneration of protoplasts severely decreased avermectin production in avermectin high-producers.

Electroporation, which is a much faster and simpler method used widely for transformation of bac-terial cells in the presence of exogenous DNA (Pigac and Schrempf 1995; Tyurin et al. 1995; Mazy-Servais et al. 1997) and for the direct transfer of plasmid DNA from a plasmid-bearing strain to plasmid-free one (Pfau and Youderian 1990; Summers and Withers 1990; Vujaklija and Davies 1995), was suggested as an alterna-tive way. In order to obtain desired results, however, it is necessary to establish the optimum conditions for a given strain, particularly in the case of industrial antibiotic-producer.

Here we aimed at determining the optimum conditions for PEG-mediated transformation of proto-plasts and electrotransformation of S. avermitilis MMR630, the industrial producer of avermectin; this is also the first report on electroduction of nonconjugative plasmid DNA between S. lividans and S. avermitilis.

MATERIALS AND METHODS

Bacterial strains and plasmids used are given in Table I.

Media. E. coli JM110 was grown in Luria–Bertani (LB) broth. S. lividans 1326 and TK24 were

grown in YEME at 30 °C; cells for the preparation of protoplasts and electrocompetent cells were grown in

*Corresponding author; present address: 300 FenLin Road, Shanghai 200 032, China.

400 WEI GONG et al. Vol. 49

YEME with 0.5 % glycine. S. avermitilis MMR630 protoplasts were regenerated in R10 – solution A (in g/L): sucrose 200, dextrin 10, yeast extract 5, casamino acids 0.1, MgCl2·6H2O 10.12, K2SO4 0.25, 2 mL trace element solution; solution B (in g/L; sterilized separately and added to A): TES buffer 5.72, proline 3, CaCl2·2H2O 2.94, NaOH 0.28, KH2PO4 0.05 (final pH 6.8). Putative transformants were purified on YMS medium (Ikeda et al. 1988) supplemented with appropriate antibiotic. YEME and double strength germina-tion medium were prepared according to Hopwood et al. (1985) except for the concentration of sucrose which was reduced to 30 % of original YEME to obtain more turbid cultures.

Table I. Strains and plasmidsa Name

Characteristics

Strains

S. avermitilis MMR630

G-1

high producer of avermectin, used for commercial production of avermectin

streptomycin-resistant derivative of MMR630, used as recipient in electroduction

wild-type strain, used as the host for plasmid pIJ702

our laboratory this work

D.A. Hopwood, John Innes Center (Norwich Research Park, Colney, UK) ditto

our laboratory Source

S. lividans 66 1326

TK24 str-6 SLP2– SLP3–, used as recipients in electroduction E. coli

JM110

dam dcm, used for the preparation and isolation of shuttle

plasmid pIJ699 for E. coli and streptomycetes

Plasmids

pIJ702

pIJ101 derivative; tsr mel Ltz–

E. Katz, John Innes Center

(Norwich Research Park, Colney, UK)

pIJ699

pIJ101 derivative; tsr vph Ltz– T. Kieser, John Innes Center

(Norwich Research Park, Colney, UK)

plasmid of S. lividans

streptomycin resistance gene thiostrepton resistance gene viomycin resistance gene

dam DNA-adenine N6-methyltransferase SLP

dcm DNA-cytosine N4-methyltransferase str Ltz lethal zygosis tsr mel tyrosinase gene vph

aAbbreviations:

Transformation of protoplasts of S. avermitilis MMR630. Spores were inoculated into 50 mL YEME in a 250-mL flask and grown for 2 d at 30 °C; then 1.5 mL was transferred to another 250-mL flask con-taining 50 mL YEME supplemented with 0.5 % glycine. After 25–30 h, mycelia were harvested and washed once with 10.3 % sucrose. The cells were suspended in 10 mL modified P medium (in g/L): sucrose 200, MgCl2·6H2O 1.22, K2SO4 0.25, 2 mL trace elements solution; added after sterilization: TES buffer 5.72, CaCl2·2H2O 0.44, KH2PO4 0.05 (final pH 6.8) with 1 mg/mL lysozyme and incubated at room temperature for 1 h with slow shaking. The resulting protoplasts were filtered through glass wool and centrifuged (1500 g, 10 min). The pellet was washed once with modified P medium, resuspended in 2 mL of the same medium, quick frozen, and stored at –70 °C.

For transformation, DNA was added to 50 μL of protoplast suspension followed by 200 μL of

T medium (40 % PEG in modified P medium, pH adjusted to 9.0) and mixed by gentle pipetting. The mix-ture was incubated at room temperature for 30 s and then quickly diluted in modified P medium. The trans-formed protoplasts were rapidly spread on R10 agar plates and incubated at 30 °C for 18 h, then the plates were overlaid with R10 soft agar with appropriate antibiotics. The plates were further incubated at the same temperature for 3–5 d.

Electrotransformation of intact cells. Cells of strain MMR630 from 50 mL of late exponential phase

culture were harvested by centrifugation (1500 g, 10 min, 4 °C) and washed thrice with ice-cold distilled wa-ter and twice with cold electroporation medium (0.5 mol/L sucrose + 1 mmol/L MgCl2). Finally, the cells were 20-fold concentrated in the same medium. Competent cells were either used directly for electroporation or stored at –70 °C. Electroporation was done using a Bio-Rad Gene PulserTM with pulse controller. Before

2004 TRANSFORMATION AND ELECTRODUCTION IN S. avermitilis 401

electroporation, 50 μL competent cells was mixed with 1 μL DNA in a 1-mm cuvette and preincubated at 50 °C for 5 min. The mixture was subjected to an electric pulse (voltage 1 kV, resistance 800 Ω, capacitance 25 μF) for various time periods and, immediately, R10 medium was added. The pulsed cells were incubated for 4–6 h at 30 °C, then plated on YMS supplemented with the appropriate antibiotic and incubated at 30 °C for 3–5 d.

Electrotransformation of protoplasts. The protoplasts were pelleted by centrifugation (1500 g,

10 min) and resuspended in 0.6 mol/L sucrose. Fifty μL protoplast solution was mixed with 1 μL DNA in an ice-cold 1-mm cuvette. After the electroporation (1 kV, 800 Ω, 25 μF), the suspension was diluted with modified P medium and spread on R10 regeneration medium. The regeneration plates were overlaid after an 18-h incubation at 30 °C with 3 mL R10 soft agar containing an antibiotic.

Electrotransformation of germlings. The spores from frozen glycerol stock were pelleted by centri-fugation (1500 g, 10 min) and washed with 1.5 mL TES buffer (50 mmol/L, pH 8). Then the pelleted spores were resuspended in 0.5 mL TES buffer and heat shocked at 50 °C for 10 min. After cooling under cold tap water, an equal volume of double strength germination medium was added and the sample was incubated at 37 °C with shaking for 2–3 h. The germinated spores were pelleted by centrifugation (1500 g, 10 min) and washed with electroporation medium (0.5 mol/L sucrose +1 mmol/L MgCl2). Germinated spores were resus-pended in the same way as intact cells.

Electroduction. The cells of donor and recipient for electroduction were prepared as mentioned

above. Twenty-five μL aliquots of the cells were mixed in a microfuge tube before being transferred to an ice-cold 1-mm cuvette. The sample was electroporated at a field strength of 10 kV/cm and the resistance of 800 ȍ, with capacitance of 25 μF. The pulsed cells were spread on R2YE (Hopwood et al. 1985) or YMS medium containing streptomycin (25 μg/mL) and thiostrepton (50 μg/mL) (only streptomycin-resistant reci-pient cells which had acquired the plasmid should grow on these plates).

RESULTS

Transformation of protoplasts

Effect of growth stage on transformation of

protoplasts. In preliminary experiments, cells were harvested at different stages from the early exponen-tial to the stationary phase and protoplasts were pre-pared. The most competent for transformation were protoplasts from the exponential phase (25–30 h cul-ture, A600 = 6–7) (Fig. 1).

Optimization of protoplast preparation. The

regeneration rate was perceptibly increased when 20 % sucrose was added to P buffer. The highest effi-ciency was obtained when Ca2+ concentration was 3 mmol/L and Mg2+ reached 6 mmol/L (Fig. 2). The

Fig. 1. Growth of S. avermitilis MMR630 (squares; A600) modified P medium gave 10–20-fold increase in trans-and the transformation efficiency (circles; n – number of formation efficiency compared with the original P me-transformants, 1/pg DNA) of protoplasts of this strain.

dium.

Various buffers with different buffering pH-

zones, including Tes, Mes and Mops were used to

determine the optimum pH. The highest regeneration efficiency (15 %) was obtained when Tes was used both in the modified P and regeneration medium. The pH optimum for protoplast regeneration was 6.8. Using Mops at the same concentration and pH, the regeneration rate decreased 3-fold.

Optimization of regeneration medium and regeneration conditions. If the standard R2YE medium

was used for protoplast regeneration in the MMR630 strain, only 10 ppm of total protoplast counts were shown to be regenerated. We therefore tested the effect of osmotic stabilizers (sucrose, mannitol, glucitol, succinic acid, NaCl) on the regeneration and improvement of the regeneration efficiency. Regeneration increased 10–20-fold when using R2YE medium supplemented with sucrose than on the same medium sup-plemented with other osmotic stabilizers; the optimum sucrose concentration was 0.5–0.6 mol/L. On modi-fied regeneration medium the maximum protoplast regeneration efficiency reached 15 %.

402 WEI GONG et al. Vol. 49

Fig. 2. Effect of Ca2+ (left) and Mg2+ (right) concentration (mmol/L) in modified P medium on the transformation efficiency (n – number of transformants, 1/pg DNA) of protoplasts of S. avermitilis MMR630.

Transformation of protoplasts. Effect of PEG type and concentration on transformation efficiency was compared in experiments with PEG-mediated transformation of the MMR630 protoplasts with pIJ702 plasmid DNA. Much better than PEG 2000 or 4000 was PEG 1000; the highest efficiency of transformation was obtained at a concentration of 40 % (W/V) (Fig. 3).

Fig. 3. Effect of PEG type (diamonds – PEG 1000; squares – PEG 2000; triangles – PEG 4000) and concentration (%) on the transformation effi-ciency (n – number of transformants, 1/pg DNA) of protoplasts of S. aver-mitilis MMR630.

Effect of protoplast concentration. In the range of protoplast concentrations between 2 × 106/μL and

2 × 108/μL the maximum efficiency (3 × 106 transformants per μg pIJ702 DNA) was obtained at a concen-tration of 108/μL (Fig. 4).

Fig. 4. Effect of protoplast concentration (c, 10/pL,i.e. 107 per μL) on the transformation efficiency (n – number of transformants, 1/pg DNA) of protoplasts ofS. avermitilis MMR630.

Fig. 5. Effect of the concentration of plasmid pIJ702DNA (ng/μL) on the transformation efficiency (n –number of transformants, 1/pg DNA) of protoplasts ofS. avermitilis MMR630.

2004 TRANSFORMATION AND ELECTRODUCTION IN S. avermitilis 403

Effect of plasmid DNA concentration. Similarly, using the range of plasmid pIJ702 DNA concen-tration of 0.004 to 16 ng/μL, the highest efficiency was obtained at a concentration of 4 ng/μL (Fig. 5).

Electrotransformation of S. avermitilis MMR630

Intact cells. Effect of the growth medium. With respect to the transformation of protoplasts, nearly no difference was observed among cells cultivated prior to protoplasting in YEME with various concentra-tions of Ca2+ or Mg2+. A remarkably different result was observed in electrotransformation which gave a 10-fold increase in efficiency after addition of 5 mmol/L CaCl2.

Effect of the electroporation medium. The presence of glycerol in electroporation medium decrea-sed significantly the electrotransformation efficiency. When this medium was prepared with 1 mmol/L MgCl2 and 0.5 mol/L sucrose, a twice higher efficiency than with 10.3 % sucrose and 15 % glycerol was obtained.

Effect of electric parameters. The most efficient electrotransformation of MMR630 intact cells with

the pIJ702 DNA was at the field strength of 10 kV/cm, 800 ȍ and pulse duration of 14–15 ms (Fig. 6). About 2 × 103 transformants per μg DNA were obtained using these conditions.

Fig. 6. Effect of pulse time (left; ms) and field strength (right; kV/cm) on the electrotrans-formation efficiency (n – number of transformants, 1/ng DNA) of S. avermitilis MMR630 with plasmid pIJ702.

Effect of the plasmid concentration. The transformation efficiency in the PEG-mediated transforma-tion remained constant in a plasmid pIJ702-concentration range of 0.004 to 4 ng/μL. The efficiency increa-sed linearly with pIJ702 DNA concentration in the range of 0.02–0.2 ng/μL (Fig. 7).

Different cell forms. Three different cell forms (germlings, protoplasts, lysozyme-treated cells) were

electrotransformed with pIJ702 DNA using the electroporation procedure. The electrotransformation of germ-lings of S. avermitilis MMR630 gave only 100 or less transformants per μg DNA. For the protoplasts, the highest efficiency was 5 × 102 per μg DNA. There was a 5-fold increase in efficiency when mycelia pre-treated with lysozyme (200 μg/mL) for 10 min at room temperature were electrotransformed (giving the efficiency of 104 transformants per μg DNA). The transformation efficiency decreased after longer time pe-riods. When testing different electric parameters, no further increase in efficiency was observed.

The electroduction of nonconjugative plasmid pIJ699 between S. avermitilis and S. lividans was

shown. The recipient strains included S. avermitilis G-1, a streptomycin-resistant mutant of strain MMR630, and S. lividans TK24 (Table II). A total of 10–100 electroductants per one electric pulse for transfer of plas-mid pIJ699 DNA was obtained between the same species (S. lividans 1326 and TK24 or S. avermitilis MMR630 and G-1). Less than 10 electoductants were obtained between different species (S. lividans 1326 and S.avermitilis G-1 or S. avermitilis MMR630 and S. lividans TK24) which demonstrated that the method is more efficient for direct transfer of nonconjugative plasmid intraspecifically than interspecifically. DISCUSSION

We demonstrated that protoplasts prepared from the mycelia grown in YEME medium to the late

exponential phase and suspended in modified P medium with 6 mmol/L Mg2+ and 3 mmol/L Ca2+ gave the

404 WEI GONG et al. Vol. 49

maximum transformation efficiency of 3 × 106 transformants per μg DNA when transformed with the plas-mid pIJ702 in the presence of 40 % PEG 1000. Matsushima and Baltz (1985) described that the addition of low levels of protamine sulfate or heterologous DNA (e.g., calf thymus DNA) to the transformation mixture substantially enhance protoplast transformation efficiency in S. fradiae and S. ambofaciens; a similar effect has not been found in S. avermitilis MMR630.

Fig. 7. Effect of pIJ702 DNA concentration (ng/μL) on the electrotrans-formation efficiency (n – number of transformants, 1/ng DNA) of S. aver-mitilis MMR630.

Table II. Efficiency of electroduction of pIJ699 between S. lividans and S. avermitilis Donor/pIJ699 (Thior Strs) Recipient (Thios Strr) Electroductant (Thior Strr)a S. lividans 1326

S. avermitilis MMR630 S. lividans 1326

S. avermitilis MMR630

S. avermitilis G-1 S. lividans TK24 S. lividans TK24 S. avermitilis G-1

1–10 10

10–100 10–100

aThio – thiostrepton, Str – streptomycin.

aNumber of colony forming units obtained after regeneration of cells from 100 μL of suspen-

sion seeded onto regeneration agar.

Brief pulses of electric fields cause the formation of pores in cell membrane and provide a local dri-ving force for ionic and molecular transport through the pores (Weaver 1990). Electroporation has been used for electrorelease of DNA in E. coli (Süleymano÷lu 2002) and for introducing of plasmid DNA into proto-plasts of Bacillus cereus (Shivarova et al. 1983), Streptococcus lactis (Harlander 1987) and also into intact cells of Lactobacillus delbrueckii (Serror et al. 2002), Streptococcus thermophilus (O’Sullivan and Fitzge-rald 1999), etc. However, the use of electroporation had only a limited effect in several Streptomyces strains (MacNeil 1987; Pigac and Schrempf 1995; Tyurin et al. 1995; Mazy-Servais et al. 1997). We showed that intact cells of S. avermitilis MMR630 harvested from a culture grown in YEME medium with 5 mmol/L CaCl2 were electrotransformed with a transformation efficiency of up to 2 × 103 per μg DNA while in ger-minated spores or protoplasts pulsed under optimized conditions the efficiency decreased to 102 per μg DNA or less. A major improvement in the efficiency was achieved in MMR630 cells by using a mild treatment with lysozyme (without protoplast formation) which gave the efficiency of 104 per μg DNA.

Electroporation as a method for introducing plasmid DNA into bacterial cells and transfer of plas-mid DNA from donor to recipient cells is considered to function both intraspecifically and interspecifically. Summers and Withers (1990) reported a simple and reliable electroporation method allowing the direct trans-fer of plasmid pBR322 between two strains of E. coli. This method was then successfully applied to the transfer of shuttle vectors between E. coli and S. typhimurium (Pfau and Youderian 1990), E. coli and Myco-bacterium sp. (Baulard et al. 1992), and E. coli and Streptomyces sp. (Vujaklija and Davies 1995). We were the first to achieve the electroduction of the nonconjugative plasmid pIJ699 between two species of S. aver-mitilis MMR630 and S. lividans TK24 by electroporation.

REFERENCES

BAULARD A., JOURDAN C., MERCENIER A., LOCHT C.: Rapid mycobacterial plasmid analysis by electroduction between Mycobacterium

spp. and Escherichia coli. Nucl.Acids Res. 20, 4105 (1992).

BIBB B.J., WARD J.M., HOPWOOD D.A.: Transformation of plasmid DNA into Streptomyces at high frequency. Nature 274, 398–400

(1978).

2004 TRANSFORMATION AND ELECTRODUCTION IN S. avermitilis 405

BURG R.W., OSTLIND D.A., CAMPBELL W.C.: Avermectins, new family of potent anthelmintic agents: producing organism and fermen-tation. Antimicrob.Agents Chemother. 15, 361–367 (1979).

HARLANDER S.K.: Transformation of Streptococcus lactis by electroporation, pp. 229–233 in J.J. Ferretti, R.C. Curtiss III (Eds): Strep-tococcal Genetics. American Society for Microbiology, Washington (DC) 1987.

HOPWOOD D.A., BIBB M.J., CHATER K.F., KIESER T., BRUTON C.J., KIESER H.M., LYDIATE D.J., SMITH C.P., WARD J.M., SCHREMPF H.:

Genetic Manipulation of Streptomyces, a Laboratory Manual. The John Innes Foundation, Norwich (UK) 1985.

HWANG Y.S., LEE J.Y., KIM E.S., CHOI C.Y.: Optimization of transformation procedures in avermectin high-producing Streptomyces

avermitilis. Biotechnol.Lett. 23, 457–462 (2001).

IKEDA H., OMURA S.: Avermectin biosynthesis. Chem.Rev. 97, 2591–2609 (1997).

IKEDA H., KOTAKI H., TANAKA H., OMURA S.: Involvement of glucose catabolism in avermectin production by Streptomyces aver-mitilis. Antimicrob.Agents Chemother. 32, 282–284 (1988).

IKEDA H., NONOMIYA T., OMURA S.: Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin

in Streptomyces avermitilis. Proc.Nat.Acad.Sci.USA 96, 9509–9514 (1999).

LEE J.Y., HWANG Y.S., KIM E.S., CHOI C.Y.: Effect of a global regulatory gene, afsR2, from Streptomyces lividans on avermectin pro-duction in Streptomyces avermitilis. J.Biosci.Bioeng. 89, 606–608 (2000).

MACNEIL D.J.: Introduction of plasmid DNA into Streptomyces lividans by electroporation. FEMS Microbiol.Lett. 42, 239–244 (1987). MACNEIL D.J., KLAPKO L.M.: Transformation of Streptomyces avermitilis by plasmid DNA. J.Ind.Microbiol. 2, 209–218 (1987). MATSUSHIMA P., BALTZ R.H.: Efficient plasmid transformation. J.Bacteriol. 163, 180–185 (1985).

MAZY-SERVAIS C., BACZKOWSKI D., DUSART J.: Electroporation of intact cells of Streptomyces parvulus and Streptomyces vinaceus.

FEMS Microbiol.Lett. 151, 135–138 (1997).

O’SULLIVAN T.F., FITZGERALD G.F.: Electrotransformation of industrial strains of Streptococcus thermophilus. J.Appl.Microbiol. 86,

275–283 (1999).

PFAU J., YOUDERIAN P.: Transferring plasmid DNA between different bacterial species with electroporation. Nucl.Acids Res. 18, 6165

(1990).

PIGAC J., SCHREMPF H.: Optimal cultural and physiological conditions for handling Streptomyces rimosus protoplasts. Appl.Environ.

Microbiol. 44, 1178–1186 (1982).

PIGAC J., SCHREMPF H.: A simple and rapid method of transformation of Streptomyces rimosus R6 and other streptomycetes by electro-poration. Appl.Environ.Microbiol. 61, 352–356 (1995).

SERROR P., SASAKI T., EHRLICH S.D., MAGUIN E.: Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. del-brueckii subsp. lactis with various plasmids. Appl.Environ.Microbiol. 68, 46–52 (2002).

SHIVAROVA N., FORSTER W., JACOB H.E., GRIGORAVA R.: Microbiological implications of electrical field effects. VII. Stimulation of

plasmid transformation of Bacillus cereus protoplasts by electric field pulses. Z.Allg.Mikrobiol. 23, 595–599 (1983).

SÜLEYMANOöLU E.: Electrorelease of Escherichia coli nucleoids. Folia Microbiol. 47, 365–370 (2002).

SUMMERS D.K., WITHERS H.L.: Electrotransfer: direct transfer of bacterial plasmid DNA by electroporation. Nucl.Acids Res. 18, 2192

(1990).

TYURIN M., STARODUBTSEVA L., LIVSHITS V.: Electrotransformation of germinating spores of Streptomyces spp. Biotech.Tech. 9, 737–

740 (1995).

VUJAKLIJA D., DAVIES J.: Direct transfer of plasmid DNA between Streptomyces spp. and E. coli by electroduction. J.Antibiot. 48, 635–

637 (1995).

WEAVER J.C.: Electroporation protocol for microorganisms, pp. 1–26 in J.A. Nicroloff (Ed.): Methods in Molecular Biology, Vol. 47.

Humana Press, Totowa (USA) 1990.