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Rapid protocol for electroporation of Clostridium perfringens
Journal of Microbiological Methods 62 (2005) 125 – 127 www.elsevier.com/locate/jmicmeth
Note
Rapid protocol for electroporation of Clostridium perfringens Alena Jira´skova´a,b, Libor Vı´tekc, Johan Feveryb, Toma´sˇ Rumld, Pavel Brannya,* a Institute of Microbiology AS CR, Vı´denˇska´ 1083, 14220 Prague 4, Czech Republic Department of Hepatology, University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, B-3000 Leuven, Belgium c 4th Department of Internal Medicine and Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Medical Faculty, Charles University of Prague, U nemocnice 2, 12808 Prague 2, Czech Republic d Department of Biochemistry and Microbiology and Center for Integrated Genomics, Institute of Chemical Technology, 166 28 Prague, Czech Republic
b
Received 13 January 2005; accepted 24 January 2005 Available online 25 February 2005
Abstract A rapid and simple method has been developed for the electroporation of Clostridium perfringens with plasmid DNA. The new improvements, harvesting cells early in the logarithmic stage of growth, keeping the cells at room temperature and the absence of post-shock incubation on ice increased transformation efficiency by one order of magnitude. D 2005 Elsevier B.V. All rights reserved. Keywords: Clostridium perfringens; Electroporation; Transformation efficiency
Clostridium perfringens is an aero-tolerant anaerobic Gram-positive bacterium, commonly found in the gastrointestinal tract of humans and animals, as well as in soil. C. perfringens can cause severe diseases in humans (McDonel, 1980) but it also is a natural part of the normal human intestinal microflora (Simon and Gorbach, 1986). Clostridia take part in the metabolism of bile acids (Wells and Hylemon, 2000) as well as in the metabolism of bilirubin (Vı´tek et al., 2000). Molecular studies in C. perfringens have been hindered by inefficient techniques for DNA manipulation in this T Corresponding author. Tel.: +42 241 062 658; fax: +42 241 722 257. E-mail address: [email protected] (P. Branny). 0167-7012/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2005.01.012
species. The major advances in C. perfringens genetics have been achieved by the development of electroporation-induced transformation methods. A notable feature of clostridial electroporation is the variety of conditions required for particular species and strains. Even though several protocols have been developed recently (Allen and Blaschek, 1990; Kim and Blaschek, 1989; Phillips-Jones, 1990; Scott and Rood, 1989), some C. perfringens strains are refractory to transformation by electroporation (Allen and Blaschek, 1990; Jira´skova´, 2004; Scott and Rood, 1989). In a previous study (Phillips-Jones, 1990) two electroporation methods were compared and modified to improve transformation efficiency of C. perfringens P90.2.2. The maximal transformation efficiency
126
A. Jira´skova´ et al. / Journal of Microbiological Methods 62 (2005) 125–127
(4.4103 transformants per microgram of DNA) was achieved when late-stationary-phase cells prepared in electoporation buffer composed of 15% glycerol were transformed with plasmid pSB92A2. In the present study we describe a rapid and simplified electroporation protocol providing higher transformation efficiency. C. perfringens P90.2.2 was a gift from Dr. Phillips-Jones, University of Leeds, UK (PhillipsJones, 1990) and was maintained in the cooked meat medium described previously (Phillips-Jones, 1995). All growth experiments were carried out anaerobically using a Gas generation kit (Oxoid, GB). Plasmid pJIR750 bearing a chloramphenicol resistance gene was a gift from Prof. J.I. Rood, Monash University, Australia (Bannam and Rood, 1993). Plasmid was maintained in Escherichia coli DH5a with selection using 30 Ag ml 1 chloramphenicol. C. perfringens transformants were selected on BHI (Oxoid) plates supplemented with 5 Ag ml 1 chloramphenicol. Plasmid DNA from E. coli was isolated by an alkaline lysis method (Sambrook et al., 1989). First, we tried electroporation of early-exponentialphase cells pretreated with lysostaphin (Scott and Rood, 1989). This procedure provided the maximal transformation efficiency that has been published so far (3.0105 transformants per microgram of DNA for C. perfringens strain 13). C. perfringens cells were treated with varying amounts of lysostaphin (0.5, 1, 10 and 20 Ag ml 1, lysostaphin-2000 units/mg; Sigma Chemicals, USA), but no transformants were obtained. In further experiments we completely omitted lysostaphin treatment, reduced the number of washes in the electroporation buffer and tested if post-electroporation incubation on ice affects transformation efficiency. All manipulations were carried out at room temperature similarly as in the original protocol (RT protocol). Alternatively, manipulations were performed at 4 8C using ice-cold electroporation buffer (Cold protocol). Ten milliliters of FTG broth (Difco Laboratories, USA) was inoculated with 0.1 ml of stock culture and incubated anaerobically overnight at 37 8C. The overnight FTG culture was used to inoculate a 100 ml of TPG (Rood et al., 1978) to a starting optical density of 0.02 (600 nm). The early-exponential-phase cell culture (OD600=0.20–0.25) was harvested by
centrifugation at 12,000g for 15 min at 20 8C, washed once in 10 ml of SMP electroporation buffer (272 mM sucrose, 7 mM sodium phosphate pH 7.4, 1 mM MgCl2) and resuspended in 10 ml of SMP. Consequently, 0.4-ml aliquots were mixed with 2 Ag of pJIR750 DNA, transferred to prechilled cuvettes (0.2 cm gap) and placed on ice for 10 min. Electroporation of cells was carried out in a Biorad Gene Pulser apparatus (Biorad Laboratories, USA) set at a capacitance of 25 AF, a resistance of 200 V and using pulses of 1.8–2.5 kV (9–12.5 kV cm 1). Immediately after the pulse delivery, half of the transformation mixture (0.2 ml) was subcultured into 10 ml of BHI regeneration broth (3.7% BHI, 0.1% sodium thioglycollate, 1.5% glucose). The second part of the transformation mixture was incubated on ice for 10 min and then transferred into 10 ml of BHI regenerating broth. Suspensions were incubated at 37 8C for 3 h to allow gene expression. Then cells were harvested by centrifugation, resuspended in 1 ml of diluent (0.37% BHI, 0.1% sodium thioglycollate) and plated either on non-selective BHI or on selective BHI agar containing 5 Ag ml 1 chloramphenicol. Alternatively, manipulations were performed at 4 8C using ice-cold electroporation buffer. The transformation efficiency was defined as the number of Cm-resistant transformants per microgram of DNA. Tables 1 and 2 show the effect of temperature (during manipulations) and applied voltage on transformation efficiency of C. perfringens P90.2.2 transformed with pJIR750 DNA. The highest transformation efficiency (1.37104) was obtained when cells were prepared at room temperature and postshock incubation on ice was omitted. Higher transformation efficiency than that achieved in the previous Table 1 Electroporation of C. perfringens P90.2.2 with pJIR750 Voltage applied (kV) 1.8 2.0 2.5
Time constant (ms) 3.2 3.3 3.2
Transformation efficiency a RT b
Icec 4
1.3710 9.03103 1.19103
5.31103 4.52103 0
Manipulations were performed at room temperature (RT protocol). Each value represents the average of three experiments. a Calculated as transformants per microgram of DNA. b Absence of post-shock incubation on ice. c Ten minutes post-shock incubation on ice.
A. Jira´skova´ et al. / Journal of Microbiological Methods 62 (2005) 125–127 Table 2 Electroporation of C. perfringens P90.2.2 with pJIR750
127
Acknowledgements
Transformation efficiency a
Voltage applied (kV)
Time constant (ms)
RT b
Icec
1.8 2.0 2.5
3.4 3.3 3.2
0.20102 0.20102 0.10102
0 0 0
Manipulations performed at 4 8C (Cold protocol). Each value represents the average of three experiments. a Calculated as transformants per microgram of DNA. b Absence of post-shock incubation on ice. c Ten minutes post-shock incubation on ice.
We thank Dr. M.K. Phillips-Jones for the gift of C. perfringens P90.2.2 and for comprehensive information concerning genetic manipulations with C. perfringens. We thank Prof. J.I. Rood for the gift of plasmid pJIR750 and for valuable advices with electroporation of lysostaphin-treated C. perfringens. This work was supported by grant GA CR 310/02/ 1436 (P.B.) and FWO G.0225.02 (J.F.).
References study (Phillips-Jones, 1990) might also have been influenced by usage of different plasmid DNA (pJIR750 vs. pSB92A2). Transformation efficiency decreased significantly (100–700 times), when electrocompetent cells were prepared at 4 8C using icecold electroporation buffer. In the case of the RT protocol, the elimination of post-shock incubation on ice resulted in up to 2.5-fold increase of the number of transformed cells, except for shocking at 2.5 kV, when cells were not transformed (Table 1). Using the Cold protocol, transformants were obtained only in the absence of post-shock incubation on ice (Table 2). In contrast with previous observations (Phillips-Jones, 1990; Scott and Rood, 1989) the highest voltage (2.5 kV) gave the lowest transformation efficiency (Table 1). It seems that such high field strength produces unacceptable levels of cell killing (99%) and is unsuitable for electroporation of C. perfringens P90.2.2. As the voltage decreased to 1.8 kV (9 kV cm 1), the transformation efficiency increased by approximately tenfold (Table 1). The ratio of cell killing was 91.8% at 1.8 kV and 96% at 2 kV. We suppose that in the case of the Cold protocol manipulations at low temperatures were the main factor affecting transformation efficiency and compromised the impact of the voltage applied. The new protocol is time-saving because earlyexponential-phase cells are transformed. The complete procedure takes approximately 6–7 h. Furthermore, no enzymatic manipulations are required. Keeping the cells at room temperature and the absence of post-shock incubation on ice significantly increased the transformation efficiency and might be applied for electroporation of previously non-transformable C. perfringens strains.
Allen, S.P., Blaschek, H.P., 1990. Factors involved in the electroporation-induced transformation of Clostridium perfringens. FEMS Microbiol. Lett. 58, 217 – 220. Bannam, T.L., Rood, J.I., 1993. Clostridium perfringens–Escherichia coli shuttle vectors that carry single antibiotic resistance determinants. Plasmid 29, 233 – 235. Jira´skova´, A., 2004. Optimization and development of genetic tools to manipulate Clostridium perfringens, one of the bilirubinreducing bacteria. Diploma thesis, Katholieke Universiteit Leuven, Leuven, Belgium. Kim, A.Y., Blaschek, H.P., 1989. Construction of an Escherichia coli–Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens. Appl. Environ. Microbiol. 55, 360 – 365. McDonel, J.L., 1980. Clostridium perfringens toxins (type A, B, C, D, E). Pharmacol. Ther. 10, 617 – 655. Phillips-Jones, M.K., 1990. Plasmid transformation of Clostridium perfringens by electroporation methods. FEMS Microbiol. Lett. 54, 221 – 226. Phillips-Jones, M.K., 1995. Introduction of recombinant DNA into Clostridium spp. Methods Mol. Biol. 47, 227 – 235. Rood, J.I., Maher, E.A., Somers, E.B., Campos, E., Duncan, C.L., 1978. Isolation and characterization of multiply antibioticresistant Clostridum perfringens strains from porcine feces. Antimicrob. Agents Chemother. 13, 871 – 880. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Scott, P.T., Rood, J.I., 1989. Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens. Gene 82, 327 – 333. Simon, G.L., Gorbach, S.L., 1986. The human intestinal microflora. Dig. Dis. Sci. 31, 147S – 162S. Vı´tek, L., Kotal, P., Jirsa, M., Malina, J., Cˇerna´, M., Chmelarˇ, D., Fevery, J., 2000. Intestinal colonization leading to fecal urobilinoid excretion may play a role in the pathogenesis of neonatal jaundice. J. Pediatr. Gastroenterol. Nutr. 30, 294 – 298. Wells, J.E., Hylemon, P.B., 2000. Identification and characterization of a bile acid 7alpha-dehydroxylation operon in Clostridium sp. strain TO-931, a highly active 7alpha-dehydroxylating strain isolated from human feces. Appl. Environ. Microbiol. 66, 1107 – 1113.
Note
Rapid protocol for electroporation of Clostridium perfringens Alena Jira´skova´a,b, Libor Vı´tekc, Johan Feveryb, Toma´sˇ Rumld, Pavel Brannya,* a Institute of Microbiology AS CR, Vı´denˇska´ 1083, 14220 Prague 4, Czech Republic Department of Hepatology, University Hospital Gasthuisberg, Catholic University of Leuven, Herestraat 49, B-3000 Leuven, Belgium c 4th Department of Internal Medicine and Institute of Clinical Biochemistry and Laboratory Diagnostics, 1st Medical Faculty, Charles University of Prague, U nemocnice 2, 12808 Prague 2, Czech Republic d Department of Biochemistry and Microbiology and Center for Integrated Genomics, Institute of Chemical Technology, 166 28 Prague, Czech Republic
b
Received 13 January 2005; accepted 24 January 2005 Available online 25 February 2005
Abstract A rapid and simple method has been developed for the electroporation of Clostridium perfringens with plasmid DNA. The new improvements, harvesting cells early in the logarithmic stage of growth, keeping the cells at room temperature and the absence of post-shock incubation on ice increased transformation efficiency by one order of magnitude. D 2005 Elsevier B.V. All rights reserved. Keywords: Clostridium perfringens; Electroporation; Transformation efficiency
Clostridium perfringens is an aero-tolerant anaerobic Gram-positive bacterium, commonly found in the gastrointestinal tract of humans and animals, as well as in soil. C. perfringens can cause severe diseases in humans (McDonel, 1980) but it also is a natural part of the normal human intestinal microflora (Simon and Gorbach, 1986). Clostridia take part in the metabolism of bile acids (Wells and Hylemon, 2000) as well as in the metabolism of bilirubin (Vı´tek et al., 2000). Molecular studies in C. perfringens have been hindered by inefficient techniques for DNA manipulation in this T Corresponding author. Tel.: +42 241 062 658; fax: +42 241 722 257. E-mail address: [email protected] (P. Branny). 0167-7012/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2005.01.012
species. The major advances in C. perfringens genetics have been achieved by the development of electroporation-induced transformation methods. A notable feature of clostridial electroporation is the variety of conditions required for particular species and strains. Even though several protocols have been developed recently (Allen and Blaschek, 1990; Kim and Blaschek, 1989; Phillips-Jones, 1990; Scott and Rood, 1989), some C. perfringens strains are refractory to transformation by electroporation (Allen and Blaschek, 1990; Jira´skova´, 2004; Scott and Rood, 1989). In a previous study (Phillips-Jones, 1990) two electroporation methods were compared and modified to improve transformation efficiency of C. perfringens P90.2.2. The maximal transformation efficiency
126
A. Jira´skova´ et al. / Journal of Microbiological Methods 62 (2005) 125–127
(4.4103 transformants per microgram of DNA) was achieved when late-stationary-phase cells prepared in electoporation buffer composed of 15% glycerol were transformed with plasmid pSB92A2. In the present study we describe a rapid and simplified electroporation protocol providing higher transformation efficiency. C. perfringens P90.2.2 was a gift from Dr. Phillips-Jones, University of Leeds, UK (PhillipsJones, 1990) and was maintained in the cooked meat medium described previously (Phillips-Jones, 1995). All growth experiments were carried out anaerobically using a Gas generation kit (Oxoid, GB). Plasmid pJIR750 bearing a chloramphenicol resistance gene was a gift from Prof. J.I. Rood, Monash University, Australia (Bannam and Rood, 1993). Plasmid was maintained in Escherichia coli DH5a with selection using 30 Ag ml 1 chloramphenicol. C. perfringens transformants were selected on BHI (Oxoid) plates supplemented with 5 Ag ml 1 chloramphenicol. Plasmid DNA from E. coli was isolated by an alkaline lysis method (Sambrook et al., 1989). First, we tried electroporation of early-exponentialphase cells pretreated with lysostaphin (Scott and Rood, 1989). This procedure provided the maximal transformation efficiency that has been published so far (3.0105 transformants per microgram of DNA for C. perfringens strain 13). C. perfringens cells were treated with varying amounts of lysostaphin (0.5, 1, 10 and 20 Ag ml 1, lysostaphin-2000 units/mg; Sigma Chemicals, USA), but no transformants were obtained. In further experiments we completely omitted lysostaphin treatment, reduced the number of washes in the electroporation buffer and tested if post-electroporation incubation on ice affects transformation efficiency. All manipulations were carried out at room temperature similarly as in the original protocol (RT protocol). Alternatively, manipulations were performed at 4 8C using ice-cold electroporation buffer (Cold protocol). Ten milliliters of FTG broth (Difco Laboratories, USA) was inoculated with 0.1 ml of stock culture and incubated anaerobically overnight at 37 8C. The overnight FTG culture was used to inoculate a 100 ml of TPG (Rood et al., 1978) to a starting optical density of 0.02 (600 nm). The early-exponential-phase cell culture (OD600=0.20–0.25) was harvested by
centrifugation at 12,000g for 15 min at 20 8C, washed once in 10 ml of SMP electroporation buffer (272 mM sucrose, 7 mM sodium phosphate pH 7.4, 1 mM MgCl2) and resuspended in 10 ml of SMP. Consequently, 0.4-ml aliquots were mixed with 2 Ag of pJIR750 DNA, transferred to prechilled cuvettes (0.2 cm gap) and placed on ice for 10 min. Electroporation of cells was carried out in a Biorad Gene Pulser apparatus (Biorad Laboratories, USA) set at a capacitance of 25 AF, a resistance of 200 V and using pulses of 1.8–2.5 kV (9–12.5 kV cm 1). Immediately after the pulse delivery, half of the transformation mixture (0.2 ml) was subcultured into 10 ml of BHI regeneration broth (3.7% BHI, 0.1% sodium thioglycollate, 1.5% glucose). The second part of the transformation mixture was incubated on ice for 10 min and then transferred into 10 ml of BHI regenerating broth. Suspensions were incubated at 37 8C for 3 h to allow gene expression. Then cells were harvested by centrifugation, resuspended in 1 ml of diluent (0.37% BHI, 0.1% sodium thioglycollate) and plated either on non-selective BHI or on selective BHI agar containing 5 Ag ml 1 chloramphenicol. Alternatively, manipulations were performed at 4 8C using ice-cold electroporation buffer. The transformation efficiency was defined as the number of Cm-resistant transformants per microgram of DNA. Tables 1 and 2 show the effect of temperature (during manipulations) and applied voltage on transformation efficiency of C. perfringens P90.2.2 transformed with pJIR750 DNA. The highest transformation efficiency (1.37104) was obtained when cells were prepared at room temperature and postshock incubation on ice was omitted. Higher transformation efficiency than that achieved in the previous Table 1 Electroporation of C. perfringens P90.2.2 with pJIR750 Voltage applied (kV) 1.8 2.0 2.5
Time constant (ms) 3.2 3.3 3.2
Transformation efficiency a RT b
Icec 4
1.3710 9.03103 1.19103
5.31103 4.52103 0
Manipulations were performed at room temperature (RT protocol). Each value represents the average of three experiments. a Calculated as transformants per microgram of DNA. b Absence of post-shock incubation on ice. c Ten minutes post-shock incubation on ice.
A. Jira´skova´ et al. / Journal of Microbiological Methods 62 (2005) 125–127 Table 2 Electroporation of C. perfringens P90.2.2 with pJIR750
127
Acknowledgements
Transformation efficiency a
Voltage applied (kV)
Time constant (ms)
RT b
Icec
1.8 2.0 2.5
3.4 3.3 3.2
0.20102 0.20102 0.10102
0 0 0
Manipulations performed at 4 8C (Cold protocol). Each value represents the average of three experiments. a Calculated as transformants per microgram of DNA. b Absence of post-shock incubation on ice. c Ten minutes post-shock incubation on ice.
We thank Dr. M.K. Phillips-Jones for the gift of C. perfringens P90.2.2 and for comprehensive information concerning genetic manipulations with C. perfringens. We thank Prof. J.I. Rood for the gift of plasmid pJIR750 and for valuable advices with electroporation of lysostaphin-treated C. perfringens. This work was supported by grant GA CR 310/02/ 1436 (P.B.) and FWO G.0225.02 (J.F.).
References study (Phillips-Jones, 1990) might also have been influenced by usage of different plasmid DNA (pJIR750 vs. pSB92A2). Transformation efficiency decreased significantly (100–700 times), when electrocompetent cells were prepared at 4 8C using icecold electroporation buffer. In the case of the RT protocol, the elimination of post-shock incubation on ice resulted in up to 2.5-fold increase of the number of transformed cells, except for shocking at 2.5 kV, when cells were not transformed (Table 1). Using the Cold protocol, transformants were obtained only in the absence of post-shock incubation on ice (Table 2). In contrast with previous observations (Phillips-Jones, 1990; Scott and Rood, 1989) the highest voltage (2.5 kV) gave the lowest transformation efficiency (Table 1). It seems that such high field strength produces unacceptable levels of cell killing (99%) and is unsuitable for electroporation of C. perfringens P90.2.2. As the voltage decreased to 1.8 kV (9 kV cm 1), the transformation efficiency increased by approximately tenfold (Table 1). The ratio of cell killing was 91.8% at 1.8 kV and 96% at 2 kV. We suppose that in the case of the Cold protocol manipulations at low temperatures were the main factor affecting transformation efficiency and compromised the impact of the voltage applied. The new protocol is time-saving because earlyexponential-phase cells are transformed. The complete procedure takes approximately 6–7 h. Furthermore, no enzymatic manipulations are required. Keeping the cells at room temperature and the absence of post-shock incubation on ice significantly increased the transformation efficiency and might be applied for electroporation of previously non-transformable C. perfringens strains.
Allen, S.P., Blaschek, H.P., 1990. Factors involved in the electroporation-induced transformation of Clostridium perfringens. FEMS Microbiol. Lett. 58, 217 – 220. Bannam, T.L., Rood, J.I., 1993. Clostridium perfringens–Escherichia coli shuttle vectors that carry single antibiotic resistance determinants. Plasmid 29, 233 – 235. Jira´skova´, A., 2004. Optimization and development of genetic tools to manipulate Clostridium perfringens, one of the bilirubinreducing bacteria. Diploma thesis, Katholieke Universiteit Leuven, Leuven, Belgium. Kim, A.Y., Blaschek, H.P., 1989. Construction of an Escherichia coli–Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens. Appl. Environ. Microbiol. 55, 360 – 365. McDonel, J.L., 1980. Clostridium perfringens toxins (type A, B, C, D, E). Pharmacol. Ther. 10, 617 – 655. Phillips-Jones, M.K., 1990. Plasmid transformation of Clostridium perfringens by electroporation methods. FEMS Microbiol. Lett. 54, 221 – 226. Phillips-Jones, M.K., 1995. Introduction of recombinant DNA into Clostridium spp. Methods Mol. Biol. 47, 227 – 235. Rood, J.I., Maher, E.A., Somers, E.B., Campos, E., Duncan, C.L., 1978. Isolation and characterization of multiply antibioticresistant Clostridum perfringens strains from porcine feces. Antimicrob. Agents Chemother. 13, 871 – 880. Sambrook, J., Fritsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Scott, P.T., Rood, J.I., 1989. Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens. Gene 82, 327 – 333. Simon, G.L., Gorbach, S.L., 1986. The human intestinal microflora. Dig. Dis. Sci. 31, 147S – 162S. Vı´tek, L., Kotal, P., Jirsa, M., Malina, J., Cˇerna´, M., Chmelarˇ, D., Fevery, J., 2000. Intestinal colonization leading to fecal urobilinoid excretion may play a role in the pathogenesis of neonatal jaundice. J. Pediatr. Gastroenterol. Nutr. 30, 294 – 298. Wells, J.E., Hylemon, P.B., 2000. Identification and characterization of a bile acid 7alpha-dehydroxylation operon in Clostridium sp. strain TO-931, a highly active 7alpha-dehydroxylating strain isolated from human feces. Appl. Environ. Microbiol. 66, 1107 – 1113.