Electroporation-Induced Transformation of Intact Cells of Clostridium ...

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Apr 18, 1988 - STEVEN P. ALLEN AND HANS P. BLASCHEK*. Department ofFood ..... We thank Bryan White for S. faecalis OG1-X and Phillip Hylemon for shuttle ... 54:268-270. 21. Shivarova, N., W. Forster, H. E. Jacob, and R. Grigorova.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1988, p. 2322-2324

Vol. 54, No. 9

0099-2240/88/092322-03$02.00/0 Copyright © 1988, American Society for Microbiology

Electroporation-Induced Transformation of Intact Cells of Clostridium perfringens STEVEN P. ALLEN AND HANS P. BLASCHEK* Department of Food Science, University of Illinois, Urbana, Illinois 61801 Received 18 April 1988/Accepted 8 June 1988

Electroporation-induced transformation of intact cells of Clostridium perfringens 3624A with plasmids pAMBl and pHR106 resulted in 3.8 x 10-5 and 4.2 x 10-4 transformants per viable cell, respectively. With respect to shuttle plasmid pHR106, these values represent a >100-fold increase in transformation frequency when compared with the values reported with polyethylene glycol-induced L-phase variants.

Polyethylene glycol-mediated plasmid transformation in Clostridium perfringens has involved the use of L-phase variants or autoplasts (11, 16, 20, 23). The problems associated with this approach include the inability of L-phase variants to revert back to the walled state (11, 16, 20), their inability to transform autoplasts with heterologous DNA derived from Escherichia coli (23), and their relatively low transformation frequency (11, 20, 23). Squires et al. (23) observed that the probability of plasmid loss per generation from L-phase variants is 100-fold greater than that from walled cells, which suggests that segregation of plasmid DNA is probably less precise during division of L-phase variants. This observation suggests that it may be difficult for plasmid DNA to become replicationally established in C. perfringens L-phase variants after transformation, and this problem may be one cause of the relatively low transformation frequencies. Another factor which affects the uptake and transfer of DNA in clostridia may be the presence of indigenous DNases associated with the cell wall and membrane (1, 14, 19). The development of an intact cell transformation procedure may overcome some of these problems. One approach for transfer of DNA into protoplasts or intact cells is electroporation. This process involves the application of a high-intensity electric field which reversibly permeabilizes the lipid bilayer membrane of the cell, thus forming a pore and permitting the entry of macromolecules (24). Initial applications of this technique involved the transformation of eucaryotic cells (10, 18). Electroporation has been used to introduce plasmid DNA into protoplasts of Bacillus cereus (21) and Streptococcus lactis (9), as well as into intact cells of Lactobacillus casei (5). The objective of the present study was to develop specific conditions and protocols for electroporation-induced transformation of intact C. perfringens cells with plasmid DNA. A plasmid-free, spontaneous rifampin- and streptomycin sulfate-resistant mutant of C. perfringens 3624A (B. C. Hampson and H. P. Blaschek, Abstr. Annu. Meet. Am. Soc. Microbiol. 1985, H108, p. 126) was utilized in this study. The stock culture was grown at 37°C in cooked-meat medium (Difco Laboratories, Detroit, Mich.) containing rifampin (2 ,ug/ml) and streptomycin (512 jig/ml) (Sigma Chemical Co., St. Louis, Mo.) and subsequently was stored in cooked-meat medium at 4°C. Selection of C. perfringens transformants was carried out by plating cells on Trypticase glucose yeast extract (TGY; 12) agar supplemented with 25 mM MgCl2, 25 mM CaCl2 (J. T. Baker Chemical Co., Phillipsburg, N.J.), *

Corresponding author.

and appropriate antibiotic. The addition of MgCl2 plus CaCl2 enhanced transformant cell recovery (data not shown). Plates were incubated under anaerobic conditions (85% N2, 10% C02, 5% H2) in an anaerobic chamber (Coy Laboratory Products, Inc., Ann Arbor, Mich.). Plasmid pAMB1 DNA was isolated from Streptococcus faecalis OG1-X by a modification of the procedure described by Clewell et al. (7). When the mid-logarithmic growth phase was attained, glycine (Sigma) was added to the culture broth to a final concentration of 3% (15). The culture was then incubated at 37°C for an additional hour before being harvested. Purified pAMB1 DNA was obtained by cesium chloride (CsCl)-ethidium bromide dye-buoyant density centrifugation of the crude lysate as described by Clewell and Smith (6, 22), except that 1 g of CsCl was added for every milliliter of sample, and 0.8 ml of a stock ethidium bromide solution (10 mg/ml) was added for every 10 ml of CsCI solution. Intact-cell electroporation-induced transformation of C. perfringens 3624A with plasmid pAMB1 was carried out with the Bio-Rad Gene Pulser (Bio-Rad Laboratories, Richmond, Calif.) set at 25 ,uF. An overnight TGY culture containing rifampin and streptomycin was used to inoculate a fresh flask of TGY which also contained rifampin and streptomycin to a starting optical density at 600 nm of 0.16. Cells were harvested (5,900 x g, 10 min, 4°C) after 1 h and 40 min of growth (optical density at 600 nm of 0.85) at 37°C, washed once in an equal volume of cold electroporationbuffer (E buffer; 0.27 M sucrose, 1 mM MgCl2, and 5 mM Na2HPO4, adjusted to pH 7.4), and suspended in 0.5 volume of cold E buffer. Cell suspension (0.8 ml) plus pAMB1 DNA (final concentration, 5 ,ug/ml) was transferred to a prechilled sample cuvette (0.4-cm gap), mixed, and allowed to sit on ice for 5 min. The sample was then shocked with the desired voltage and placed on ice for 5 min. A sample (0.3 ml) was serially diluted in E buffer and plated on nonselective TGY agar to determine the percent kill. The remaining 0.5 ml was added to 2.5 ml of TGY-expression medium (TGY containing 25 mM CaCl2, 25 mM MgCl2, and 0.075% agar) and incubated for 3 h at 37°C before being plated on TGYselective agar containing 25 ,ug of erythromycin (Sigma) per ml. Plates were incubated anaerobically for 48 h before transformants were counted. The effect of applied voltage on intact-cell electroporationinduced transformation of C. perfringens 3624A with plasmid pAMB1 is presented in Table 1. As the voltage was increased to the maximum of the Gene Pulser (2.5 kV), the transformation efficiency and percent kill also increased. 2322

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TABLE 1. Effect of applied voltage on intact-cell electroporation-induced transformation of C. perfringens 3624A with plasmid pAMBla Voltage

Time constant

Transformation

(kV)

(ms)b

efficiencyc

% K Ild

1.0 1.5 2.0 2.5

8.7 8.3 6.9 6.0

0 5.4 6.5 x 101 1.4 x 102

6.4 47.4 77.8 81.8

a Values represent the averages of duplicate determinations. A transformation frequency of 3.8 x 10'5 transformants per viable cell was obtained after electroporation at 2.5 kV. b Decay time from peak voltage to approximately 37% of peak voltage. c Calculated as transformants per microgram of DNA. Selection was carried out on TGY containing 25 ,ug of erythromycin per ml. d 1 - (viable cells after shock/total input cells) x 100.

It is important to incubate the shocked cells for 3 h at 37°C in TGY-expression medium to obtain transformants. This period of time may be necessary to allow repair of cellular damage caused by electric shock and to allow expression of the plasmid DNA-encoded antibiotic resistance. Controls for each transformation experiment consisted of an unshocked cell culture without added plasmid DNA, an unshocked cell culture containing added plasmid DNA, and a shocked cell culture without added plasmid DNA. Emr colonies were not recovered from the control TGY-selective agar plates. The presence of plasmid pAMB1 in C. perfringens Emr transformants was confirmed by a plasmid isolation protocol obtained from B. C. Hampson (personal communication). The procedure was as follows. (i) Late-log-phase cells were harvested (5,900 x g, 4°C, 15 min), washed once in 0.1 volume of TES-A-sucrose buffer (150 mM NaCl, 50 mM Tris-hydrochloride, 5 mM EDTA, 25% sucrose, [pH 7.4]), and suspended in 0.1 volume of Tris-sucrose buffer (4). (ii) The cell suspension was subjected to a lysozyme treatment (final concentration, 2 mg/ml) for 5 min at 37°C and placed on ice for 25 min. (iii) EDTA (0.25 M) was added to a final concentration of 10 mM before addition of the alkaline lysis buffer (8). (iv) A deproteinization was carried out following NaCl precipitation of the chromosomal DNA by a phenol, phenol-chloroform (1:1), and chloroform extraction procedure (17). Sucrose was added to the wash buffer and Trissucrose was used as the suspension buffer to prevent autolysis and release of cell-wall compartmentalized DNase during the isolation of plasmid DNA from C. perfringens (1, 2). Purified plasmid was obtained by CsCI-ethidium bromide dye-buoyant density centrifugation, as described above. HindlIl restriction endonuclease digestion of pAMB1 plasmid DNA isolated from C. perfringens and S. faecalis OG1-X was performed as described by the manufacturer (Bethesda Research Laboratories, Gaithersburg, Md.). Digested plasmid DNA was subjected to agarose gel electrophoresis with a 1.2% SeaKem GTG-agarose gel (FMC Corp., Rockland, Maine). Electrophoresis was carried out in Tris-acetate running buffer (13) at 40 V (constant voltage) for 16 h with a horizontal flatbed slab gel apparatus (model H4; Bethesda Research). DNA bands were visualized and photographed as described by Blaschek and Solberg (3). DNA fragments were transferred to a nitrocellulose filter (Bethesda Research) essentially as described by Maniatis et al. (17). Modifications included the use of neutralization buffer consisting of 3.0 M NaCl, 0.5 M Tris-hydrochloride (pH 7.9), and 20x SSC (lx SSC is 0.15 M NaCI plus 0.015 M sodium citrate) in the blotting tray. S. faecalis-derived linearized pAMB1, obtained after di-

FIG. 1. Southern blot hybridization of [32P]dCTP-labeled linearized pAMB1 to HindIII-digested pAMB1 isolated from either S. faecalis (lane 1) or C. perfringens (lane 2) transformants. A total of 14 DNA fragments ranged in size from 4.1 to 0.5 kilobases (given on the right).

gestion with AvaI, was labeled with [a-32P]dCTP (Du Pont, NEN Research Products, Wilmington, Del.) by nick translation (Bethesda Research). The labeled probe was separated from unincorporated radioactive nucleotide with Elutip-d as described by the manufacturer (Schleicher & Schuell, Inc., Keene, N.H.). The specific activity of the probe was determined by counting a 10-,ul sample in an LS 9000 liquid scintillation counter (Beckman Instruments, Inc., Palo Alto, Calif.). Southern blot filter hybridization and autoradiography were performed as described by Maniatis et al. (17), with the following modifications: (i) prehybridization and hybridization fluids did not contain sodium dodecyl sulfate or EDTA, and (ii) prehybridization was carried out for 12 to 16 h, while hybridization was carried out for 24 h. The specific activity of the pAMBl probe was determined to be 2.5 x 108 cpm/,ug

of DNA. It was added to the hybridization solution to give a final concentration of 107 cpm/ml. The results of Southern blot hybridization of [32P]dCTP-labeled linearized pAMB1 to HindIII-digested pAMB1 isolated from either S. faecalis or C. perfringens transformants are presented in Fig. 1. Identical fragment banding patterns with demonstrated homology between the probe and restricted plasmids isolated from either C. perfringens or S. faecalis indicate that the electroporation-induced transformation process as well as maintenance in C. perfringens does not result in structural modification of pAMB1. Plasmid pHR106, a 7.9-kilobase shuttle plasmid (20), was

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also used to transform C. perfringens 3624A. Electroporation-induced transformation was performed as previously described except that only one voltage setting was used (2.5 kV) and transformants were selected on TGY agar containing 20 ,ug of chloramphenicol (Sigma) per ml. Plasmid pHR106 was transformed into C. perfringens at an efficiency of 1.2 x 103 transformants per jig of DNA and a frequency of 4.2 x 10-4 transformants per viable cell after electroporation. This plasmid transformation frequency for pHR16 is over 100-fold better than the frequency reported for L-phase variant transformation systems (11, 20, 23). Shuttle plasmid pHR106 demonstrated increased efficiency and frequency of electroporation-induced transforrnation when compared with pAMB1. This improved transformability may be explained by either the difference in size between pAMBl (26.5 kilobases) and pHR106 (7.9 kilobases) or the fact that pHR106 contains a clostridial origin of replication. Plasmid transformation of C. perfringens is no longer restricted to the use of L-phase variants or autoplasts. Successful transformation of intact C. perfringens cells with plasmids pAMB1 and pHR106 was accomplished by electroporation. This system of gene transfer overcomes the problems of the reversion of L-phase variants to the walled state, low transformation frequency due to improper segregation of plasmid DNA during division of L-phase variants, and the potential interaction of plasmid DNA with DNase associated with the cell wall or membrane. Experiments are currently under way to optimize the transformation frequencies reported here. This work was supported in part by New Investigator Research Award PHS 5 R23 A122417 from the National Institutes of Health to H.P.B., by Hatch grant 50-304 from the University of Illinois Agricultural Experiment Station, and by grant 1-2-69157 from the University of Illinois Research Board. We thank Bryan White for S. faecalis OG1-X and Phillip Hylemon for shuttle plasmid pHR106. LITERATURE CITED P., and M. A. Klacik. 1984. Role of DNase in recovery of plasmid DNA from Clostridium perfringens. Appl. Environ. Microbiol. 48:178-181. Blaschek, H. P., and M. A. Klacik. 1985. Development of a cell wash buffer that minimizes nucleic acid loss from Clostridium perfringens 10543 A. Can. J. Microbiol. 31:575-578. Blaschek, H. P., and M. Solberg. 1981. Isolation of a plasmid responsible for caseinase activity in Clostridium perfringens ATCC 3626B. J. Bacteriol. 147:262-266. Brefort, G., M. Magot, H. lonesco, and M. Sebald. 1977. Characterization and transferability of Clostridium perfringens plasmids. Plasmid 1:52-66. Chassy, B. M., and J. L. Flickinger. 1987. Transformation of Lactobacillus casei by electroporation. FEMS Microbiol. Lett. 44:173-177. Clewell, D. B. 1972. Nature of ColE, plasmid replication in Escherichia coli in the presence of chloramphenicol. J. Bacteriol. 110:667-676. Clewell, D. B., Y. Yagi, G. M. Dunny, and S. K. Schultz. 1974. Characterization of three plasmid deoxyribonucleic acid molecules in a strain of Streptococcus faecalis: identification of a

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