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RNA transfection of Escherichia coli by electroporation
Biochimica et Biophysica Acta. 1007 (1989) 127-129
127
Elsevier BBA 90129
BBA Repo~
RNA transfecfion of Escherichia coil by electroporation Akira Taketo Department of Biochemistry L Fukui Medical School. Fukui (Japan)
(Received 26 August1988) Key words: Electroporation;Voltageshock; RNA transfection:PhageRNA; ( E. co/i) Cells of EschericMa coli were efficiently transfected with QD phage RNA by electropo~tion. A single voltage shock at 6.25 k V / e m with a 25 p F capacitor resulted in an infectivity yield considerably higher than that attained by a lysozyme-EDTA spheroplast method or a CaCI 2 procedure. A linear relationship was found between concentration of the input RNA and yield of the transfectants, over a wide range. Efficiency of the electroporation-mediated transfection (electrotransfectton) was increased by addition of certain sodium salts but decreased by preineubation in a Tr|s buffer containing sucrose.
By brief exposure to a high electric field strength, cellular or viral nucleic acids can successfuly be introduced into various eukaryotic cells [1-5]. This voltage shock method, also referred to as electroporation or electropermeabilization, has recently been applied to several bacterial species including Streptococcus lactis [6], Lactobacillus casei [7], Campylobacterjejuni [8], and Escherichia coil [9], for transformation with recombiaant plasmids or transfection with viral DNAs. In contrast to DNA, RNA transfects CaCI2-treated E. coil cells less efficiently [10], and lysozyme-EDTA (LE) spheroplasts have been exclusively used for assay of its infectivity. The spheroplasts are, however, fragile and deficient in colonyforming ability, and their competence fluctuates considerably, depending on the preparation. Another disadvantage is liberation of RNase activity into the surrounding medium during and after lysozyme-EDTA treatment. In order to overcome these difficulties and to study the transfer and expression of foreign RNA molecules in bacteria, electroporationmediated RNA transfection was tested in intact cells of E. coll.
The RNA of Qfl phage was extracted with phenol from the virus particles partially purified by differential centrifugation. Strains of E. coil used were K12A(F +) and C(F-). The bacteria were grown in L broth supplemented with 0.08% glucose, at 37 or 44 o C with shaking. When the A660 of the culture reached 0.6, cells were collected, washed with chilled 20 mM Tris-HCl buffer (pH 7.5) and suspended in the buffer at an A66o of 40.
Con'espondence: A. Taketo, Department of Biochemistry 1. Fukui Medical School, Matsuoka, Fukui910-11,Japan.
To the chilled suspension, 1/50 voi. of RNA in 10 mM Tris-HCi buffer (pH 7.5) was added and the RNA-containing suspension was mixed with an equal volume of chilled 105[ sucrose. Where specified, sodium citrate, sodium phosphate and sodium sulfate (each I raM) were added together with sucrose. The mixture (0.8 ml) was #petted into a Bio-Rad electroporation cuvette with an interelectrode distance of 0.4 cm, and subjected to an electric shock (set voltage 2.5 kV; initial field strength 6.25 kV/cm; set capacitance 25 ~F) as previously described [9], using a Bio-Rad Gene Pulser TM apparatus. Each sample was then diluted with chilled 10 mM Tris-HCl buffer (pH 7.5) containing 5% sucrose and plated with the indicator cells (E. coli K12A(F+)). Procedures for the CaCl2-dependent transfection were essentially similar to those described earlier [11]. In certain cases, bacteria grown at 44°C were used and concentration of the competent cell suspension was doubled (A66o--30). The heat pulse, where indicated, was performed at 42°C for 1 min followed by quenching in ice/water. LE-spheroplasts were prepared and infected as described previously [12]. Values are shown as averages for at least three determinations. Under the electroporation conditions developed for DNA transfection and transformation, cell of E. coli K12A(F +) were effectively transfected with Qfl RNA. The transfectants were not produced at initial field strength of less than 0.625 kV/cm or at capacitance below 0.25 #F. When sodium salts of citrate, phosphate and sulfate (each 1 raM) were added to the eleetroporalion medium, infectivity yield of the RNA increased about 3-fold (Table l). In E. coli C (F °) also, similar results were obtained with the RNA transfections. Besides the salt effect, the electroporation-mediated trans-
0167-4781/89/$03.50 © 1989ElsevierScience PublishersB.V.(BiomedicalDivision)
128 TABLE 1
-[
Comparison of efflciencies of transfection by electroporation. CaCl2 syston and spheroplast mcthad Strain KI2A(F + ) grown at the indicated temperature was used for preparation of the recipient cells. An electrical impul~ was delivered at a field strength of 6.25 kV/cm, with a 25 FF capacitor, in the presence or absence of sodium citrate, phosphate and sulfate. After k ~ at 0 ° C for 10 rain, plaque-forming unit (PFU) was titrated, For Caaa-dependent chilled competent cell suspension (Aem -15) was mixed with 1/2 volume of RNA and kept at 0 ° C for 20 rain. Infectivity yield was d e ~ directly or after a heat pulse at 42°C for 1 rain. The spheroplast infectivity was assayed after 12 rain incubation at 37 o C in L broth containing 20% sucrose.
mmsfmio~
Method
Culture
Condition
temp, ( e C)
Yield of transfectants (PFU/ml)
EIc~troporation
37 37 44
standard salts added salts added
1.|, 106 3.1.106 1.6.106
CaCI~
37 37 44 44
without heat pulse with heat pulse without heat pulse with heat pulse
6.3.10 ~ 2.0.10 ~ 1.0-10 4 3,8,10 ~
in 20~ sucrose/ L broth
6.2.10 4
LE,spheroplasts 37
fer of DNA was promoted by growing the recipient cells at high temperatures such as ~ ° C (unpublished data). In the cells grown at M°C, however, addition of the sodium salts did not improve the transfection efficiency of RNA as well as of DNA. The infectivity yield of Qfl RNA was 170- to 490-times higher in electroporation than in the CaCl2.treated cells. (To the CaCI2-dependent transfection of RNA, heat pulse was detrimental. In additional, infectivity of the RNA was rather reduped, when 2-fold-concentrated recipient cells were used,) For RNA transfection, the LE, spheroplast method was about 10-times more efficient than the CaCI~ procedure, Compared to the electropermeabilized bacteria, LE, sphemplasts were less competent for Qp RNA: yield of the transf~tants was 20- to 50-times hi~er in the electroporation. TABLE II £~
Ofl ~ ~ o ~
oN the efficiency of RNA transfection by electro-
Immediately after addition of QB RNA, KIZA(F + ) cell suspension in chilled 20 ram Ttis, HC! buffer (pH 7,5) was mixed at 0 o C with an equal volume of 10~ sucrose. At the indicated time, aliquot of the mixture was subjected to el~troporation (6.25 kV/cm. 25/AF)and the ,~ r , ,
P t e i n g ~ t i o n time
(rain) 1 $ 10
Yield of transfectants
(PFU/ml)
(%)
6.8-10 e 3.7-10 e l.g- 10e
100 54 28
t
'1('""" "'
!
" "|
I
6 o 6
?, ~ 3 2
R N A
(pg/ml)
Fig. i. Relationship between Q~8 RNA concentration and the plaque yield. Varying amounts of Q~ RNA were added to E. cdi KI2A(F + ) cells suspended in chilled 20 mM Tris.HCI (pH 7.$). After mixing with an equal volume of chilled 10% sucrose containing sodium salts of citrate, phosphate and sulfate (each 2 mM), the samples were subjected to electroporation under the standard conditions.
To evaluate the assay system, the yield of transfectants should be in proportion to the input RNA. Varying amounts of RNA were therefore added to the recipient bacteria at a constant density, in the same volume, then mixed with chilled sucrose solution containing the three sodium salts, and subjected to a single voltage shock at 6.25 kV/cm using a 25 ttF capacitor. The resistance-capacitance time constant was in a range of 2.9 to 3.1 ms. The result of plaque counting is illustrated in Fig. 1. It is evident that the yield of transfectants increases in parallel with the amount of the added RNA from 0.001 to 100 pg/mi. In the experiments described above, the RNA-cell mixture was subjected to electroporation after incubation for 10 min at 0 ° C in 10 mM Tris-HCI buffer (pH 7.5) containing 5~ sucrose. Although this pretreatment improves efficiency and reproducibility of the electroporationmediated DNA transfer (unpublished data), the effect on the RNA transfection is unknown. To test the influence of preincubation, the RNA-containing cell suspension in chilled 20 mM Tris-HCl buffer (pH 7.5) was mixed at 0°C with an equal volume of 10% sucrose and, at intervals, aliquots of the mixture were subjected to electroporation. Unlike DNA transfection, the yield of transfectants was high when the gNA-containing cell suspension was electroporated immediately after mixing with sucrose: under our conditions 1.4.10 s transfectants were produced per microgram crude RNA. Electroporation after a 10 min pretreatment caused 72%
129
loss in infectivity yield. Whether cellular RNase activity is involved or not in this decrease remains to be elucidated. In contrast to the efficient transfer of DNA, introduction of RNA molecules into E. coli cells is more difficult [10]. Even in spheroplasts, the transfection efficiency of RNA is 10-2 to 10-3 of that of microvirid DNA [13]. Electroporation is superior to conventional RNA transfection methods for E. coli, in its higher efficiency as well as in applicability to intact cells. The efficiency may further be increased by modifying electric parameters and by using RNase-deficient cells. In addition to phage-RNA transfection, this simple method may be applied for introduction and expression of messenger RNA or tRNA in bacteria. References I Neumann, E., Schaefer-Ridder. M., Wang, Y. and Hofschneider, P.H. 0982) EMBO J. 1,841-845.
2 Potter, H,, Weir, L. and Leder, P. (1984) Proc. Natl. Acad. Sci. USA 81, 7161-7165. 3 Hashimoto, H., Morikawa, H., Yamada, Y. and Kimura, A. 0985) Appl. Microbiol. Biotechnol. 21, 336-339. 4 Morikawa, H,, iida, A., Matsui, C., lkegami, M. and Yamada, Y. (1986) Gene 41, 121-124. 5 Gibson, W.C., White, T.C., Laird, P.W. and Borst, P. (1987) EMBO J. 6, 2457-2461. 6 Harlander, S.K. (1986) in Streptococcal Genetics (Ferretti, J.J. and Curtiss, R.C., eds.), pp. 229-233, ASM Publications, Washington, DC. 7 Chassy, B.M. and Flickinger, J.L. (1987) FEMS Microbiol. Lett. 44, 173-177. 8 Miller, J.F., Dower, W.J. and Tompkins, L.S. (1988) Proc. Na*.l. Acad. Sci, USA 85, 856-860. 9 Taketo, A. (1988) Biochim. Biophys. Acta 949, 318-324. 10 Taketo, A. (1972) J. Biochem. 72, 973-979. II Taketo, A. (1982) Z. Naturforsch. 37c, 87-92. 12 Taketo, A., Yasuda, S. and Sekiguchi, M. ~1972) J. Mol. Biol. 70~ !-14. 13 Hofschneider, P.H. and Delius, H. (1968) Methods Enzymol. 12. 880~886.
127
Elsevier BBA 90129
BBA Repo~
RNA transfecfion of Escherichia coil by electroporation Akira Taketo Department of Biochemistry L Fukui Medical School. Fukui (Japan)
(Received 26 August1988) Key words: Electroporation;Voltageshock; RNA transfection:PhageRNA; ( E. co/i) Cells of EschericMa coli were efficiently transfected with QD phage RNA by electropo~tion. A single voltage shock at 6.25 k V / e m with a 25 p F capacitor resulted in an infectivity yield considerably higher than that attained by a lysozyme-EDTA spheroplast method or a CaCI 2 procedure. A linear relationship was found between concentration of the input RNA and yield of the transfectants, over a wide range. Efficiency of the electroporation-mediated transfection (electrotransfectton) was increased by addition of certain sodium salts but decreased by preineubation in a Tr|s buffer containing sucrose.
By brief exposure to a high electric field strength, cellular or viral nucleic acids can successfuly be introduced into various eukaryotic cells [1-5]. This voltage shock method, also referred to as electroporation or electropermeabilization, has recently been applied to several bacterial species including Streptococcus lactis [6], Lactobacillus casei [7], Campylobacterjejuni [8], and Escherichia coil [9], for transformation with recombiaant plasmids or transfection with viral DNAs. In contrast to DNA, RNA transfects CaCI2-treated E. coil cells less efficiently [10], and lysozyme-EDTA (LE) spheroplasts have been exclusively used for assay of its infectivity. The spheroplasts are, however, fragile and deficient in colonyforming ability, and their competence fluctuates considerably, depending on the preparation. Another disadvantage is liberation of RNase activity into the surrounding medium during and after lysozyme-EDTA treatment. In order to overcome these difficulties and to study the transfer and expression of foreign RNA molecules in bacteria, electroporationmediated RNA transfection was tested in intact cells of E. coll.
The RNA of Qfl phage was extracted with phenol from the virus particles partially purified by differential centrifugation. Strains of E. coil used were K12A(F +) and C(F-). The bacteria were grown in L broth supplemented with 0.08% glucose, at 37 or 44 o C with shaking. When the A660 of the culture reached 0.6, cells were collected, washed with chilled 20 mM Tris-HCl buffer (pH 7.5) and suspended in the buffer at an A66o of 40.
Con'espondence: A. Taketo, Department of Biochemistry 1. Fukui Medical School, Matsuoka, Fukui910-11,Japan.
To the chilled suspension, 1/50 voi. of RNA in 10 mM Tris-HCi buffer (pH 7.5) was added and the RNA-containing suspension was mixed with an equal volume of chilled 105[ sucrose. Where specified, sodium citrate, sodium phosphate and sodium sulfate (each I raM) were added together with sucrose. The mixture (0.8 ml) was #petted into a Bio-Rad electroporation cuvette with an interelectrode distance of 0.4 cm, and subjected to an electric shock (set voltage 2.5 kV; initial field strength 6.25 kV/cm; set capacitance 25 ~F) as previously described [9], using a Bio-Rad Gene Pulser TM apparatus. Each sample was then diluted with chilled 10 mM Tris-HCl buffer (pH 7.5) containing 5% sucrose and plated with the indicator cells (E. coli K12A(F+)). Procedures for the CaCl2-dependent transfection were essentially similar to those described earlier [11]. In certain cases, bacteria grown at 44°C were used and concentration of the competent cell suspension was doubled (A66o--30). The heat pulse, where indicated, was performed at 42°C for 1 min followed by quenching in ice/water. LE-spheroplasts were prepared and infected as described previously [12]. Values are shown as averages for at least three determinations. Under the electroporation conditions developed for DNA transfection and transformation, cell of E. coli K12A(F +) were effectively transfected with Qfl RNA. The transfectants were not produced at initial field strength of less than 0.625 kV/cm or at capacitance below 0.25 #F. When sodium salts of citrate, phosphate and sulfate (each 1 raM) were added to the eleetroporalion medium, infectivity yield of the RNA increased about 3-fold (Table l). In E. coli C (F °) also, similar results were obtained with the RNA transfections. Besides the salt effect, the electroporation-mediated trans-
0167-4781/89/$03.50 © 1989ElsevierScience PublishersB.V.(BiomedicalDivision)
128 TABLE 1
-[
Comparison of efflciencies of transfection by electroporation. CaCl2 syston and spheroplast mcthad Strain KI2A(F + ) grown at the indicated temperature was used for preparation of the recipient cells. An electrical impul~ was delivered at a field strength of 6.25 kV/cm, with a 25 FF capacitor, in the presence or absence of sodium citrate, phosphate and sulfate. After k ~ at 0 ° C for 10 rain, plaque-forming unit (PFU) was titrated, For Caaa-dependent chilled competent cell suspension (Aem -15) was mixed with 1/2 volume of RNA and kept at 0 ° C for 20 rain. Infectivity yield was d e ~ directly or after a heat pulse at 42°C for 1 rain. The spheroplast infectivity was assayed after 12 rain incubation at 37 o C in L broth containing 20% sucrose.
mmsfmio~
Method
Culture
Condition
temp, ( e C)
Yield of transfectants (PFU/ml)
EIc~troporation
37 37 44
standard salts added salts added
1.|, 106 3.1.106 1.6.106
CaCI~
37 37 44 44
without heat pulse with heat pulse without heat pulse with heat pulse
6.3.10 ~ 2.0.10 ~ 1.0-10 4 3,8,10 ~
in 20~ sucrose/ L broth
6.2.10 4
LE,spheroplasts 37
fer of DNA was promoted by growing the recipient cells at high temperatures such as ~ ° C (unpublished data). In the cells grown at M°C, however, addition of the sodium salts did not improve the transfection efficiency of RNA as well as of DNA. The infectivity yield of Qfl RNA was 170- to 490-times higher in electroporation than in the CaCl2.treated cells. (To the CaCI2-dependent transfection of RNA, heat pulse was detrimental. In additional, infectivity of the RNA was rather reduped, when 2-fold-concentrated recipient cells were used,) For RNA transfection, the LE, spheroplast method was about 10-times more efficient than the CaCI~ procedure, Compared to the electropermeabilized bacteria, LE, sphemplasts were less competent for Qp RNA: yield of the transf~tants was 20- to 50-times hi~er in the electroporation. TABLE II £~
Ofl ~ ~ o ~
oN the efficiency of RNA transfection by electro-
Immediately after addition of QB RNA, KIZA(F + ) cell suspension in chilled 20 ram Ttis, HC! buffer (pH 7,5) was mixed at 0 o C with an equal volume of 10~ sucrose. At the indicated time, aliquot of the mixture was subjected to el~troporation (6.25 kV/cm. 25/AF)and the ,~ r , ,
P t e i n g ~ t i o n time
(rain) 1 $ 10
Yield of transfectants
(PFU/ml)
(%)
6.8-10 e 3.7-10 e l.g- 10e
100 54 28
t
'1('""" "'
!
" "|
I
6 o 6
?, ~ 3 2
R N A
(pg/ml)
Fig. i. Relationship between Q~8 RNA concentration and the plaque yield. Varying amounts of Q~ RNA were added to E. cdi KI2A(F + ) cells suspended in chilled 20 mM Tris.HCI (pH 7.$). After mixing with an equal volume of chilled 10% sucrose containing sodium salts of citrate, phosphate and sulfate (each 2 mM), the samples were subjected to electroporation under the standard conditions.
To evaluate the assay system, the yield of transfectants should be in proportion to the input RNA. Varying amounts of RNA were therefore added to the recipient bacteria at a constant density, in the same volume, then mixed with chilled sucrose solution containing the three sodium salts, and subjected to a single voltage shock at 6.25 kV/cm using a 25 ttF capacitor. The resistance-capacitance time constant was in a range of 2.9 to 3.1 ms. The result of plaque counting is illustrated in Fig. 1. It is evident that the yield of transfectants increases in parallel with the amount of the added RNA from 0.001 to 100 pg/mi. In the experiments described above, the RNA-cell mixture was subjected to electroporation after incubation for 10 min at 0 ° C in 10 mM Tris-HCI buffer (pH 7.5) containing 5~ sucrose. Although this pretreatment improves efficiency and reproducibility of the electroporationmediated DNA transfer (unpublished data), the effect on the RNA transfection is unknown. To test the influence of preincubation, the RNA-containing cell suspension in chilled 20 mM Tris-HCl buffer (pH 7.5) was mixed at 0°C with an equal volume of 10% sucrose and, at intervals, aliquots of the mixture were subjected to electroporation. Unlike DNA transfection, the yield of transfectants was high when the gNA-containing cell suspension was electroporated immediately after mixing with sucrose: under our conditions 1.4.10 s transfectants were produced per microgram crude RNA. Electroporation after a 10 min pretreatment caused 72%
129
loss in infectivity yield. Whether cellular RNase activity is involved or not in this decrease remains to be elucidated. In contrast to the efficient transfer of DNA, introduction of RNA molecules into E. coli cells is more difficult [10]. Even in spheroplasts, the transfection efficiency of RNA is 10-2 to 10-3 of that of microvirid DNA [13]. Electroporation is superior to conventional RNA transfection methods for E. coli, in its higher efficiency as well as in applicability to intact cells. The efficiency may further be increased by modifying electric parameters and by using RNase-deficient cells. In addition to phage-RNA transfection, this simple method may be applied for introduction and expression of messenger RNA or tRNA in bacteria. References I Neumann, E., Schaefer-Ridder. M., Wang, Y. and Hofschneider, P.H. 0982) EMBO J. 1,841-845.
2 Potter, H,, Weir, L. and Leder, P. (1984) Proc. Natl. Acad. Sci. USA 81, 7161-7165. 3 Hashimoto, H., Morikawa, H., Yamada, Y. and Kimura, A. 0985) Appl. Microbiol. Biotechnol. 21, 336-339. 4 Morikawa, H,, iida, A., Matsui, C., lkegami, M. and Yamada, Y. (1986) Gene 41, 121-124. 5 Gibson, W.C., White, T.C., Laird, P.W. and Borst, P. (1987) EMBO J. 6, 2457-2461. 6 Harlander, S.K. (1986) in Streptococcal Genetics (Ferretti, J.J. and Curtiss, R.C., eds.), pp. 229-233, ASM Publications, Washington, DC. 7 Chassy, B.M. and Flickinger, J.L. (1987) FEMS Microbiol. Lett. 44, 173-177. 8 Miller, J.F., Dower, W.J. and Tompkins, L.S. (1988) Proc. Na*.l. Acad. Sci, USA 85, 856-860. 9 Taketo, A. (1988) Biochim. Biophys. Acta 949, 318-324. 10 Taketo, A. (1972) J. Biochem. 72, 973-979. II Taketo, A. (1982) Z. Naturforsch. 37c, 87-92. 12 Taketo, A., Yasuda, S. and Sekiguchi, M. ~1972) J. Mol. Biol. 70~ !-14. 13 Hofschneider, P.H. and Delius, H. (1968) Methods Enzymol. 12. 880~886.