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Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579
Journal of Microbiological Methods 67 (2006) 543 – 548 www.elsevier.com/locate/jmicmeth
Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579 Nathalie Turgeon a , Christian Laflamme a , Jim Ho b , Caroline Duchaine a,c,⁎ a Institut universitaire de cardiologie et de pneumologie, Hôpital Laval, Université Laval, Quebec City, Québec, Canada Biological Detection Group, Defense Research and Development Canada Suffield, CFB Suffield, Ralston, Alberta, Canada Département de biochimie et microbiologie, Faculté des sciences et de génie, Université Laval, Quebec City, Québec, Canada b
c
Received 22 March 2006; received in revised form 16 May 2006; accepted 16 May 2006 Available online 3 July 2006
Abstract An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 × 109 cfu μg− 1 ml− 1 with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 × 106 and 1 × 108 cfu μg− 1 ml− 1. The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 103 times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 106 times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols. © 2006 Elsevier B.V. All rights reserved. Keywords: Bacillus cereus; Electroporation; Glycine; Threonine; Wall-weakening
1. Introduction Bacillus cereus is a Gram-positive sporulating bacterium commonly found in soil and air. It is also an opportunistic pathogen that can induce food poisoning (Schoeni and Wong, 2005). Among the B. cereus group, B. cereus is closely related to Bacillus thuringiensis, Bacillus mycoides and Bacillus anthracis, the causative agent of anthrax (Ash et al., 1991).
⁎ Corresponding author. Centre de recherche, Hôpital Laval, 2725 Chemin Ste-Foy, Québec, Canada G1V 4G5. Tel.: +1 418 656 8711x5837; fax: +1 418 656 4509. E-mail address: [email protected] (C. Duchaine). 0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2006.05.005
Genetic manipulations are useful for various types of studies and applications (Sambrook and Russell, 2001). DNA can be incorporated into Bacillus cells using several techniques (cf. review by Bron and Vehmaanperä, 1990). Electroporation is quick and simple compared to other methods, and results are highly reproducible. However, among Bacillus species, different electroporation efficiencies have been reported due to the large variability between methods (Bron and Vehmaanperä, 1990). In recent years, efforts have been made to increase success rates up to 106 cfu μg− 1 for Bacillus subtilis using a combination of osmoprotective agents and high electric fields (Xue et al., 1999). Similar findings have been reported for Bacillus pseudofirmus using osmolarity protection followed by treatment with
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N. Turgeon et al. / Journal of Microbiological Methods 67 (2006) 543–548
glycine and high electric field (Ito and Nagane, 2001). These strategies have not been tested for members of B. cereus group. For B. cereus, the highest electroporation efficiency is 103 cfu μg− 1, being insufficient for many applications such as mutagenesis (Belliveau and Trevors, 1989). In the present study, we evaluated the influence of modulating growth cycle, electric field and amino-acid composition (glycine and threonine) to elaborate an electro-transformation procedure of B. cereus ATCC 14579.
2.2. Preparation of electro-competent cells B. cereus ATCC14579 cells growth was monitored by measurement of optical density at 600 nm using a GeneQuant pro UV/Vis spectrophotometer (Biochrom Ltd, Cambridge England). When wall-weakening treatment was performed, 5% glycine or DL-threonine and 250 mM sucrose were added during different stages of growth and incubated 1 h, 2 h or 3 h, 37 °C, 200 rpm. Cells were washed five times in electroporation buffer (250 mM sucrose, 1 mM Hepes, 1 mM MgCl2, 10% glycerol, pH 7.0) and concentrated 150-fold.
2. Materials and methods 2.3. Electroporation 2.1. Bacterial strains, plasmids and media Bacterial strains used in this study are Escherichia coli MC1061 (Wertman et al., 1986), B. cereus ATCC14579, B. cereus HER-1399 and B. cereus HER1414. E. coli MC1061 was grown in Luria broth (Difco Laboratories, Detroit, MI) at 37 °C, 200 rpm. B. cereus was grown at 37 °C, 200 rpm in trypticase soy broth (TSB) (Difco Laboratories) or on trypticase soy agar (TSA) (Difco Laboratories). When appropriate, 100 μg/ ml of ampicilin or 20 μg/ml of tetracycline, chloramphenicol or erythromycin (Sigma-Aldrich, Oakville, Ontario, Canada) were added. Plasmids used in this study are listed in Table 1. Plasmidic DNA of pLS1 and pAMβ1 was purified from E. coli MC1061 using Qiagen plasmid maxi kit (Qiagen, Chatsworth, CA). For the isolation of pT181 from Staphylococcus aureus and pC194 form of B. subtilis, the purification procedure was preceded by a lysostaphin treatment (25 ng/ml, 15 min, 37 °C) and a lysozyme treatment (10 mg/ml lysozyme, 30 min, 37 °C), respectively.
Table 1 Plasmids used in this study Plasmid pC194
Relevant characteristics
Staphylococcus aureus, Cmr, 2.9 kb pLS1 Streptococcus agalactiae, pMV158 Δmob derivative, Tcr, 4.4 kb pMTL500Eres Enterococcus faecalis, pAMβ1 derivative, Ampr, Emr, 7.1 kb pT181 Staphylococcus aureus, Tcr, 4.4 kb
References (Horinouchi and Weisblum, 1982) (Lacks et al., 1986)
Electroporation was performed at 25 μF using a Bio-Rad Gene Pulser apparatus (Bio-Rad laboratories) equipped with a Bio-Rad pulse controller. Electroporation was carried out in 2 mm cuvettes (Bio-Rad laboratories, Richmond, CA) where 100 μl cells were combined with 1 μg DNA (voltage range up to 12.5 kV cm− 1) or in 1 mm electroporation cuvettes with 50 μl cells combined with 500 ng plasmid, in order to respect cells/DNA proportions (voltage range between 13 and 25 kV cm− 1). After electroporation, cell suspensions were diluted with 1 ml of TSB supplemented with 250 mM sucrose, 5 mM MgCl2, 5 mM MgSO4 and incubated for 2 h at 37 °C, 200 rpm to allow expression of antibiotic resistance markers. Aliquots were spread onto tryptic soy agar (TSA, Difco Laboratories) supplemented with appropriate antibiotic. Transformants harboring antibiotic resistance were counted following overnight incubation. 2.4. Statistical analyses The statistical analyses were carried out with Statistical Analytical Software (SAS). Results were expressed as mean value ± standard deviation. Data were analyzed using paired t-test. All reported p-values were declared significant at p < 0.05. 3. Results and discussion 3.1. Optimization of growth conditions
(Swinfield et al., 1991) (Khan and Novick, 1983)
Ampr, ampicilin resistance; Cmr, chloramphenicol resistance; Emr, erythromycin resistance; Tcr, tetracycline resistance.
We transformed late-stage cultures of B. cereus with pLS1 using the electroporation protocol described by Vehmaanperä (Bron and Vehmaanperä, 1990). The transformation efficiency was 103 cfu μg− 1 ml− 1 and is in agreement with published findings.
N. Turgeon et al. / Journal of Microbiological Methods 67 (2006) 543–548
Glycine was used to enhance electro-competence of B. cereus as previously described (Hammes et al., 1973). For many species, the presence of the wallweakening agent glycine during bacterial growth improves the transformation rate (Framson et al., 1997; Helmark et al., 2004; Lee et al., 2002; McDonald et al., 1995). Glycine is incorporated into interpeptide bridges of peptidoglycan instead of L- and D-alanine thereby reducing the extent of cross-linking of the wall (Hammes et al., 1973). Unfortunately, in the presence of glycine bacterial growth is highly variable (Buckley et al., 1999; Dunny et al., 1991). To circumvent this problem, glycine was added to an exponentially growing culture in order to maximize the effects without compromising cell growth. This method has previously been used for cryotransformation of B. anthracis (Stepanov et al., 1990) and electroporation of lactic acid bacteria, streptococci and B. pseudofirmus (Buckley et al., 1999; Ito and Nagane, 2001; Mason et al., 2005; Turgeon and Moineau, 2001). Wall-weakening treatments strongly affect the growth of B. cereus. In 10% glycine, total cellular lysis occurred within 1 h even if sucrose was added as isotonic agent. With 5% glycine, the OD remained nearly stable for at least an hour (data not shown). To determine the optimal growth stage and incubation time suitable for B. cereus, glycine and sucrose were added during different stages of growth (Fig. 1). Cells were incubated for 0 h, 1 h, 2 h, or 3 h with glycine prior to washing in electroporation buffer and electroporation with pLS1 DNA, at 12.5 kV cm− 1, 200 Ω. For most of the tested conditions (Fig. 1), cells collected in early-stage gave better electroporation rates
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Fig. 2. Optimization of electroporation parameters. Relationship between numbers of transformants obtained per microgram of pLS1 DNA and per milliliter of competent cells and voltage. Electroporations were performed in early growth stage cultures (O.D. 0.2–0.4) incubated for 1 h with glycine, at the voltages indicated and at resistance levels of 100 Ω (○), 200 Ω (□) or 400 Ω (△).
than late-stage culture. Treatment with glycine for 1 or 2 h provided similar data; however, with 3 h glycine treatment results were highly variable due to cell lysis in the samples. Under these experimental conditions, optimal results (3 × 105 cfu μg− 1 ml− 1) were obtained in early growth stage culture (O.D. 0.2–0.5) at 1 h incubation with glycine (Fig. 1). 3.2. Optimization of electroporation conditions Electroporation parameters were further optimized as follows. Experiments were performed with pLS1 DNA using the optimal growth parameters defined in the previous section (O.D. 0.2–0.4 with 1 h incubation in glycine–sucrose). Electroporation parameters were set as shown in Fig. 2. The best transformation efficiency was obtained using high electric field at 200 Ω, 20 kV cm− 1. Transformation efficiency declined markedly at higher voltages. Using this procedure, a transformation efficiency of 9 × 105 cfu μg− 1 ml− 1 was obtained for B. cereus. 3.3. Comparison with other studies
Fig. 1. Relationship between numbers of transformants obtained per microgram of pLS1 DNA and per milliliter of competent cells and cell density. Cells were grown until the optical density indicated was obtained and then further incubated with glycine for 0 h (○), 1 h (□), 2 h (△), and 3 h (◊). Electroporations were performed with low electric field (200 Ω, 12.5 kV cm− 1).
To validate the procedure and to compare results with other studies, transformation with plasmid pC194 was tested. This plasmid has the same replication mechanism and a similar size than pUB110 (McKenzie et al., 1986), the plasmid used in B. subtilis and B. pseudofirmus where transformation efficiencies of 106 cfu μg− 1 have been achieved. The transformation rate obtained using our procedure (O.D. 0.2–0.5, 1 h incubation with 5% glycine, 20 kV cm− 1, 200 Ω) was 5 × 108 cfu μg− 1 ml− 1
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Fig. 3. Comparison of different wall-weakening treatments with untreated cells. Transformation rates obtained with pC194 DNA under high electric field (200 Ω, 20 kV cm− 1) with 3 different batches (○, □, △) of untreated cells (control), glycine treated, and DL-threonine treated cells. Standard deviation and p-value are displayed on the graphic.
with pC194 plasmid DNA (compared to 9 × 105 cfu μg− 1 ml− 1 with pLS1 DNA). This result is 100 times greater than that obtained in earlier studies with other Bacillus sp. using osmolarity protection, glycine treatment, high electric field and pUB110 DNA (Ito and Nagane, 2001; Xue et al., 1999). Bacterial growth during the 2 h of recovery after electroporation cannot be responsible of this enhancement because a 2 h or 3 h recovery was used in previous studies (Ito and Nagane, 2001; McDonald et al., 1995; Xue et al., 1999). 3.4. Comparison of wall-weakening treatments Previous studies document that DL-threonine is an effective wall-weakening agent for B. subtilis (McDonald et al., 1995). We compared the effects of DLthreonine and glycine as described above. Furthermore, to determine the influence of wall-weakening treatment on electroporation rates, replicates of the procedure were done and statistical analyses were performed. Cells were grown until OD 0.2–0.4 was reached and collected without treatment or incubated 1 h with 5% glycine or DL-threonine and 250 mM sucrose. OD was monitored
during wall-weakening treatment to ensure that cellular lysis did not occur. Experiments were done using three different cell batches. Electroporation treatments were performed three to six times for each cell lot as described previously, with pC194 plasmid DNA and high electric field (25 μF, 20 kV cm− 1, 200 Ω). The greatest transformation efficiencies were obtained in the absence of wall-weakening agent (control) and were up to 2 × 109 cfu μg− 1 ml− 1 compared with 5 × 108 cfu μg− 1 ml− 1 for glycine treatment and 2 × 108 cfu μg− 1 ml− 1 for DL-threonine treatment (Fig. 3). To our knowledge, this level of electro-competence is the highest ever achieved for the Bacillus genus. Paired t-test performed on data from all experimental conditions documented a significant and reproducible difference (p < 0.05). Under these conditions, DL-threonine reduced the transformation rates of B. cereus by 50% compared to glycine treatment, and glycine reduced the transformability by 71% compared to untreated cells. Wall-weakening treatment did not improve transformation rates with pLS1 DNA either (data not shown). Thus, it is apparent that under weak electric field, glycine positively affects transformation rate (Fig. 1). With high electric field, the presence of wall-weakening agents decreases electro-transformability of B. cereus (Fig. 3). As such, glycine and DLthreonine treatment does not confer a positive effect on transformation rate or reduce the overall burden of work. Consequently, we would recommend the use of the electroporation protocol without addition of wallweakening agents. 3.5. Transformation efficiencies with various strains plasmids Many factors influence transformation efficiency, namely, size and replication mechanism of the plasmid used. We compared the electroporation efficiencies obtained with four different plasmids, harboring various size, selection marker, copy number per cell and replication mechanism (Table 2). The highest results were obtained with the smallest plasmid
Table 2 Transformation efficiencies obtained with various plasmids Plasmids pC194 pLS1 pT181 pMTL500Eres
Plasmid copy number
Replication mechanism
Size (kb)
Antibiotic selection
Transformation efficiencies
Medium Low High Low
RC RC RC θ
2.9 4.4 4.4 7.1
Cm Tc Tc Em
8 × 108 2 × 106 8 × 106 1 × 108
θ, theta replication mechanism; Cm, chloramphenicol; Em, erythromycin; RC, rolling circle replication mechanism; Tc, tetracycline.
N. Turgeon et al. / Journal of Microbiological Methods 67 (2006) 543–548
harboring a chloramphenicol selection marker (pC194). High electroporation efficiency was also obtained with the larger plasmid carrying an erythromycin resistance gene (pMTL500Eres). The lowest transformation rates were obtained with plasmids containing tetracycline resistance marker. In this case, for the same plasmid size, the plasmid with a high copy number (pT181) gave better results than the plasmid with a low copy number (pLS1). Those results showed that chloramphenicol and erythromycin are more suitable selection markers for the electro-transformation of B. cereus. With the appropriate selection marker, the described electroporation protocol gave high transformation efficiencies, even for large theta replicating plasmids with low copy number per cell (Table 2). Interesting results were also obtained with two other strains of B. cereus (6 × 106 cfu μg− 1 ml− 1 with HER1399 and 3 × 106 cfu μg− 1 ml− 1 with HER-1414 with pC194 DNA). Those results are 103 times superior than previously reported for B. cereus strains. Therefore, this protocol can increase the electro-transformation efficiencies for several strains of B. cereus, but growth and electroporation parameters should be adapted to each specific strain, as previously reported for other bacterial species (Buckley et al., 1999). This paper describes an electro-transformation method for B. cereus ATCC14579 with results of 2 × 109 cfu μg− 1 ml− 1; this level of transformation is the highest ever reported for Bacillus sp. The method is quick, simple, highly reproducible, appropriate for many plasmids and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols. In addition, this procedure may be useful for other Bacillus strains that are refractory to electroporation. Acknowledgements We are very grateful to Gloria del Solar, MarieFrançoise Noirot-Gros, Sleem Khan and Laurent Jannière for the generous provision of the pLS1, pC194, pT181 and pMTL500Eres plasmids. We also acknowledge Felix d'Hérelle Reference Center for Bacterial Viruses for providing B. cereus strains HER1399 and HER-1414. We thank the members of the Groupe de Recherche en Santé Respiratoire for helpful discussions. NT is the recipient of a postdoctoral fellowship from the Groupe de Recherche en Santé Respiratoire. CD acknowledges a CIHR/IRSST scholarship. This work was funded by Defense Research and Development Canada Suffield.
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References Ash, C., Farrow, J.A., Dorsch, M., Stackebrandt, E., Collins, M.D., 1991. Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int. J. Syst. Bacteriol. 41, 343–346. Belliveau, B.H., Trevors, J.T., 1989. Transformation of Bacillus cereus vegetative cells by electroporation. Appl. Environ. Microbiol. 55, 1649–1652. Bron, S., Vehmaanperä, J., 1990. Electrotransformation of B. amyloliquefaciens/B. subtilis. In: Harwood, C., Cutting, S. (Eds.), Molecular Biological Methods for Bacillus. John Wiley and sons, Chichester, pp. 156–157. Buckley, N.D., Vadeboncoeur, C., LeBlanc, D.J., Lee, L.N., Frenette, M., 1999. An effective strategy, applicable to Streptococcus salivarius and related bacteria, to enhance or confer electroporation competence. Appl. Environ. Microbiol. 65, 3800–3804. Dunny, G.M., Lee, L.N., LeBlanc, D.J., 1991. Improved electroporation and cloning vector system for Gram-positive bacteria. Appl. Environ. Microbiol. 57, 1194–1201. Framson, P.E., Nittayajarn, A., Merry, J., Youngman, P., Rubens, C.E., 1997. New genetic techniques for group B streptococci: highefficiency transformation, maintenance of temperature-sensitive pWV01 plasmids, and mutagenesis with Tn917. Appl. Environ. Microbiol. 63, 3539–3547. Hammes, W., Schleifer, K.H., Kandler, O., 1973. Mode of action of glycine on the biosynthesis of peptidoglycan. J. Bacteriol. 116, 1029–1053. Helmark, S., Hansen, M.E., Jelle, B., Sorensen, K.I., Jensen, P.R., 2004. Transformation of Leuconostoc carnosum 4010 and evidence for natural competence of the organism. Appl. Environ. Microbiol. 70, 3695–3699. Horinouchi, S., Weisblum, B., 1982. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150, 815–825. Ito, M., Nagane, M., 2001. Improvement of the electro-transformation efficiency of facultatively alkaliphilic Bacillus pseudofirmus OF4 by high osmolarity and glycine treatment. Biosci. Biotechnol. Biochem. 65, 2773–2775. Khan, S.A., Novick, R.P., 1983. Complete nucleotide sequence of pT181, a tetracycline-resistance plasmid from Staphylococcus aureus. Plasmid 10, 251–259. Lacks, S., Lopez, P., Greenberg, B., Espinosa, M., 1986. Identification and analysis of genes for tetracycline resistance and replication functions in the broad-host-range plasmid pLS1. J. Mol. Biol. 192, 753–765. Lee, S.H., Cheung, M., Irani, V., Carroll, J.D., Inamine, J.M., Howe, W.R., Maslow, J.N., 2002. Optimization of electroporation conditions for Mycobacterium avium. Tuberculosis 82, 167–174. Mason, C.K., Collins, M.A., Thompson, K., 2005. Modified electroporation protocol for lactobacilli isolated from the chicken crop facilitates transformation and the use of a genetic tool. J. Microbiol. Methods 60, 353–363. McDonald, I.R., Riley, P.W., Sharp, R.J., McCarthy, A.J., 1995. Factors affecting the electroporation of Bacillus subtilis. J. Appl. Bacteriol. 79, 213–218. McKenzie, T., Hoshino, T., Tanaka, T., Sueoka, N., 1986. The nucleotide sequence of pUB110: some salient features in relation to replication and its regulation. Plasmid 15, 93–103. Sambrook, J., Russell, D.W., 2001. Molecular Cloning: A Laboratory Manual, 3 edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
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Schoeni, J.L., Wong, A.C., 2005. Bacillus cereus food poisoning and its toxins. J. Food Prot. 68, 636–648. Stepanov, A.S., Puzanova, O.B., Dityatkin, S., Loginova, O.G., Ilyashenko, B.N., 1990. Glycine-induced cryotransformation of plasmids into Bacillus anthracis. J. Gen. Microbiol. 136, 1217–1221. Swinfield, T.J., Janniere, L., Ehrlich, S.D., Minton, N.P., 1991. Characterization of a region of the Enterococcus faecalis plasmid pAM beta 1 which enhances the segregational stability of pAM beta 1-derived cloning vectors in Bacillus subtilis. Plasmid 26, 209–221.
Turgeon, N., Moineau, S., 2001. Isolation and characterization of a Streptococcus thermophilus plasmid closely related to the pMV158 family. Plasmid 45, 171–183. Wertman, K.F., Wyman, A.R., Botstein, D., 1986. Host/vector interactions which affect the viability of recombinant phage lambda clones. Gene 49, 253–262. Xue, G.-P., Johnson, J.S., Dalrymple, B.P., 1999. High osmolarity improves the electro-transformation efficiency of the Grampositive bacteria Bacillus subtilis and Bacillus licheniformis. J. Microbiol. Methods 34, 183–191.
Elaboration of an electroporation protocol for Bacillus cereus ATCC 14579 Nathalie Turgeon a , Christian Laflamme a , Jim Ho b , Caroline Duchaine a,c,⁎ a Institut universitaire de cardiologie et de pneumologie, Hôpital Laval, Université Laval, Quebec City, Québec, Canada Biological Detection Group, Defense Research and Development Canada Suffield, CFB Suffield, Ralston, Alberta, Canada Département de biochimie et microbiologie, Faculté des sciences et de génie, Université Laval, Quebec City, Québec, Canada b
c
Received 22 March 2006; received in revised form 16 May 2006; accepted 16 May 2006 Available online 3 July 2006
Abstract An electro-transformation procedure was established for Bacillus cereus ATCC 14579. Using early growth-stage culture and high electric field, the ectroporation efficiency was up to 2 × 109 cfu μg− 1 ml− 1 with pC194 plasmid DNA. The procedure was tested with three other plasmids, of various sizes, replication mechanisms and selection markers, and the transformation efficiencies ranged between 2 × 106 and 1 × 108 cfu μg− 1 ml− 1. The effects of two wall-weakening agents on electroporation rates were also evaluated. The transformation rate that was reached with our procedure is 103 times higher than that previously obtained with members of the Bacillus genus with similar plasmids, and 106 times superior than that achieved with available protocols for B. cereus. The proposed method is quick, simple, efficient with small rolling circle plasmids and large theta replicating plasmids with low copy number per cell, and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols. © 2006 Elsevier B.V. All rights reserved. Keywords: Bacillus cereus; Electroporation; Glycine; Threonine; Wall-weakening
1. Introduction Bacillus cereus is a Gram-positive sporulating bacterium commonly found in soil and air. It is also an opportunistic pathogen that can induce food poisoning (Schoeni and Wong, 2005). Among the B. cereus group, B. cereus is closely related to Bacillus thuringiensis, Bacillus mycoides and Bacillus anthracis, the causative agent of anthrax (Ash et al., 1991).
⁎ Corresponding author. Centre de recherche, Hôpital Laval, 2725 Chemin Ste-Foy, Québec, Canada G1V 4G5. Tel.: +1 418 656 8711x5837; fax: +1 418 656 4509. E-mail address: [email protected] (C. Duchaine). 0167-7012/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2006.05.005
Genetic manipulations are useful for various types of studies and applications (Sambrook and Russell, 2001). DNA can be incorporated into Bacillus cells using several techniques (cf. review by Bron and Vehmaanperä, 1990). Electroporation is quick and simple compared to other methods, and results are highly reproducible. However, among Bacillus species, different electroporation efficiencies have been reported due to the large variability between methods (Bron and Vehmaanperä, 1990). In recent years, efforts have been made to increase success rates up to 106 cfu μg− 1 for Bacillus subtilis using a combination of osmoprotective agents and high electric fields (Xue et al., 1999). Similar findings have been reported for Bacillus pseudofirmus using osmolarity protection followed by treatment with
544
N. Turgeon et al. / Journal of Microbiological Methods 67 (2006) 543–548
glycine and high electric field (Ito and Nagane, 2001). These strategies have not been tested for members of B. cereus group. For B. cereus, the highest electroporation efficiency is 103 cfu μg− 1, being insufficient for many applications such as mutagenesis (Belliveau and Trevors, 1989). In the present study, we evaluated the influence of modulating growth cycle, electric field and amino-acid composition (glycine and threonine) to elaborate an electro-transformation procedure of B. cereus ATCC 14579.
2.2. Preparation of electro-competent cells B. cereus ATCC14579 cells growth was monitored by measurement of optical density at 600 nm using a GeneQuant pro UV/Vis spectrophotometer (Biochrom Ltd, Cambridge England). When wall-weakening treatment was performed, 5% glycine or DL-threonine and 250 mM sucrose were added during different stages of growth and incubated 1 h, 2 h or 3 h, 37 °C, 200 rpm. Cells were washed five times in electroporation buffer (250 mM sucrose, 1 mM Hepes, 1 mM MgCl2, 10% glycerol, pH 7.0) and concentrated 150-fold.
2. Materials and methods 2.3. Electroporation 2.1. Bacterial strains, plasmids and media Bacterial strains used in this study are Escherichia coli MC1061 (Wertman et al., 1986), B. cereus ATCC14579, B. cereus HER-1399 and B. cereus HER1414. E. coli MC1061 was grown in Luria broth (Difco Laboratories, Detroit, MI) at 37 °C, 200 rpm. B. cereus was grown at 37 °C, 200 rpm in trypticase soy broth (TSB) (Difco Laboratories) or on trypticase soy agar (TSA) (Difco Laboratories). When appropriate, 100 μg/ ml of ampicilin or 20 μg/ml of tetracycline, chloramphenicol or erythromycin (Sigma-Aldrich, Oakville, Ontario, Canada) were added. Plasmids used in this study are listed in Table 1. Plasmidic DNA of pLS1 and pAMβ1 was purified from E. coli MC1061 using Qiagen plasmid maxi kit (Qiagen, Chatsworth, CA). For the isolation of pT181 from Staphylococcus aureus and pC194 form of B. subtilis, the purification procedure was preceded by a lysostaphin treatment (25 ng/ml, 15 min, 37 °C) and a lysozyme treatment (10 mg/ml lysozyme, 30 min, 37 °C), respectively.
Table 1 Plasmids used in this study Plasmid pC194
Relevant characteristics
Staphylococcus aureus, Cmr, 2.9 kb pLS1 Streptococcus agalactiae, pMV158 Δmob derivative, Tcr, 4.4 kb pMTL500Eres Enterococcus faecalis, pAMβ1 derivative, Ampr, Emr, 7.1 kb pT181 Staphylococcus aureus, Tcr, 4.4 kb
References (Horinouchi and Weisblum, 1982) (Lacks et al., 1986)
Electroporation was performed at 25 μF using a Bio-Rad Gene Pulser apparatus (Bio-Rad laboratories) equipped with a Bio-Rad pulse controller. Electroporation was carried out in 2 mm cuvettes (Bio-Rad laboratories, Richmond, CA) where 100 μl cells were combined with 1 μg DNA (voltage range up to 12.5 kV cm− 1) or in 1 mm electroporation cuvettes with 50 μl cells combined with 500 ng plasmid, in order to respect cells/DNA proportions (voltage range between 13 and 25 kV cm− 1). After electroporation, cell suspensions were diluted with 1 ml of TSB supplemented with 250 mM sucrose, 5 mM MgCl2, 5 mM MgSO4 and incubated for 2 h at 37 °C, 200 rpm to allow expression of antibiotic resistance markers. Aliquots were spread onto tryptic soy agar (TSA, Difco Laboratories) supplemented with appropriate antibiotic. Transformants harboring antibiotic resistance were counted following overnight incubation. 2.4. Statistical analyses The statistical analyses were carried out with Statistical Analytical Software (SAS). Results were expressed as mean value ± standard deviation. Data were analyzed using paired t-test. All reported p-values were declared significant at p < 0.05. 3. Results and discussion 3.1. Optimization of growth conditions
(Swinfield et al., 1991) (Khan and Novick, 1983)
Ampr, ampicilin resistance; Cmr, chloramphenicol resistance; Emr, erythromycin resistance; Tcr, tetracycline resistance.
We transformed late-stage cultures of B. cereus with pLS1 using the electroporation protocol described by Vehmaanperä (Bron and Vehmaanperä, 1990). The transformation efficiency was 103 cfu μg− 1 ml− 1 and is in agreement with published findings.
N. Turgeon et al. / Journal of Microbiological Methods 67 (2006) 543–548
Glycine was used to enhance electro-competence of B. cereus as previously described (Hammes et al., 1973). For many species, the presence of the wallweakening agent glycine during bacterial growth improves the transformation rate (Framson et al., 1997; Helmark et al., 2004; Lee et al., 2002; McDonald et al., 1995). Glycine is incorporated into interpeptide bridges of peptidoglycan instead of L- and D-alanine thereby reducing the extent of cross-linking of the wall (Hammes et al., 1973). Unfortunately, in the presence of glycine bacterial growth is highly variable (Buckley et al., 1999; Dunny et al., 1991). To circumvent this problem, glycine was added to an exponentially growing culture in order to maximize the effects without compromising cell growth. This method has previously been used for cryotransformation of B. anthracis (Stepanov et al., 1990) and electroporation of lactic acid bacteria, streptococci and B. pseudofirmus (Buckley et al., 1999; Ito and Nagane, 2001; Mason et al., 2005; Turgeon and Moineau, 2001). Wall-weakening treatments strongly affect the growth of B. cereus. In 10% glycine, total cellular lysis occurred within 1 h even if sucrose was added as isotonic agent. With 5% glycine, the OD remained nearly stable for at least an hour (data not shown). To determine the optimal growth stage and incubation time suitable for B. cereus, glycine and sucrose were added during different stages of growth (Fig. 1). Cells were incubated for 0 h, 1 h, 2 h, or 3 h with glycine prior to washing in electroporation buffer and electroporation with pLS1 DNA, at 12.5 kV cm− 1, 200 Ω. For most of the tested conditions (Fig. 1), cells collected in early-stage gave better electroporation rates
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Fig. 2. Optimization of electroporation parameters. Relationship between numbers of transformants obtained per microgram of pLS1 DNA and per milliliter of competent cells and voltage. Electroporations were performed in early growth stage cultures (O.D. 0.2–0.4) incubated for 1 h with glycine, at the voltages indicated and at resistance levels of 100 Ω (○), 200 Ω (□) or 400 Ω (△).
than late-stage culture. Treatment with glycine for 1 or 2 h provided similar data; however, with 3 h glycine treatment results were highly variable due to cell lysis in the samples. Under these experimental conditions, optimal results (3 × 105 cfu μg− 1 ml− 1) were obtained in early growth stage culture (O.D. 0.2–0.5) at 1 h incubation with glycine (Fig. 1). 3.2. Optimization of electroporation conditions Electroporation parameters were further optimized as follows. Experiments were performed with pLS1 DNA using the optimal growth parameters defined in the previous section (O.D. 0.2–0.4 with 1 h incubation in glycine–sucrose). Electroporation parameters were set as shown in Fig. 2. The best transformation efficiency was obtained using high electric field at 200 Ω, 20 kV cm− 1. Transformation efficiency declined markedly at higher voltages. Using this procedure, a transformation efficiency of 9 × 105 cfu μg− 1 ml− 1 was obtained for B. cereus. 3.3. Comparison with other studies
Fig. 1. Relationship between numbers of transformants obtained per microgram of pLS1 DNA and per milliliter of competent cells and cell density. Cells were grown until the optical density indicated was obtained and then further incubated with glycine for 0 h (○), 1 h (□), 2 h (△), and 3 h (◊). Electroporations were performed with low electric field (200 Ω, 12.5 kV cm− 1).
To validate the procedure and to compare results with other studies, transformation with plasmid pC194 was tested. This plasmid has the same replication mechanism and a similar size than pUB110 (McKenzie et al., 1986), the plasmid used in B. subtilis and B. pseudofirmus where transformation efficiencies of 106 cfu μg− 1 have been achieved. The transformation rate obtained using our procedure (O.D. 0.2–0.5, 1 h incubation with 5% glycine, 20 kV cm− 1, 200 Ω) was 5 × 108 cfu μg− 1 ml− 1
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Fig. 3. Comparison of different wall-weakening treatments with untreated cells. Transformation rates obtained with pC194 DNA under high electric field (200 Ω, 20 kV cm− 1) with 3 different batches (○, □, △) of untreated cells (control), glycine treated, and DL-threonine treated cells. Standard deviation and p-value are displayed on the graphic.
with pC194 plasmid DNA (compared to 9 × 105 cfu μg− 1 ml− 1 with pLS1 DNA). This result is 100 times greater than that obtained in earlier studies with other Bacillus sp. using osmolarity protection, glycine treatment, high electric field and pUB110 DNA (Ito and Nagane, 2001; Xue et al., 1999). Bacterial growth during the 2 h of recovery after electroporation cannot be responsible of this enhancement because a 2 h or 3 h recovery was used in previous studies (Ito and Nagane, 2001; McDonald et al., 1995; Xue et al., 1999). 3.4. Comparison of wall-weakening treatments Previous studies document that DL-threonine is an effective wall-weakening agent for B. subtilis (McDonald et al., 1995). We compared the effects of DLthreonine and glycine as described above. Furthermore, to determine the influence of wall-weakening treatment on electroporation rates, replicates of the procedure were done and statistical analyses were performed. Cells were grown until OD 0.2–0.4 was reached and collected without treatment or incubated 1 h with 5% glycine or DL-threonine and 250 mM sucrose. OD was monitored
during wall-weakening treatment to ensure that cellular lysis did not occur. Experiments were done using three different cell batches. Electroporation treatments were performed three to six times for each cell lot as described previously, with pC194 plasmid DNA and high electric field (25 μF, 20 kV cm− 1, 200 Ω). The greatest transformation efficiencies were obtained in the absence of wall-weakening agent (control) and were up to 2 × 109 cfu μg− 1 ml− 1 compared with 5 × 108 cfu μg− 1 ml− 1 for glycine treatment and 2 × 108 cfu μg− 1 ml− 1 for DL-threonine treatment (Fig. 3). To our knowledge, this level of electro-competence is the highest ever achieved for the Bacillus genus. Paired t-test performed on data from all experimental conditions documented a significant and reproducible difference (p < 0.05). Under these conditions, DL-threonine reduced the transformation rates of B. cereus by 50% compared to glycine treatment, and glycine reduced the transformability by 71% compared to untreated cells. Wall-weakening treatment did not improve transformation rates with pLS1 DNA either (data not shown). Thus, it is apparent that under weak electric field, glycine positively affects transformation rate (Fig. 1). With high electric field, the presence of wall-weakening agents decreases electro-transformability of B. cereus (Fig. 3). As such, glycine and DLthreonine treatment does not confer a positive effect on transformation rate or reduce the overall burden of work. Consequently, we would recommend the use of the electroporation protocol without addition of wallweakening agents. 3.5. Transformation efficiencies with various strains plasmids Many factors influence transformation efficiency, namely, size and replication mechanism of the plasmid used. We compared the electroporation efficiencies obtained with four different plasmids, harboring various size, selection marker, copy number per cell and replication mechanism (Table 2). The highest results were obtained with the smallest plasmid
Table 2 Transformation efficiencies obtained with various plasmids Plasmids pC194 pLS1 pT181 pMTL500Eres
Plasmid copy number
Replication mechanism
Size (kb)
Antibiotic selection
Transformation efficiencies
Medium Low High Low
RC RC RC θ
2.9 4.4 4.4 7.1
Cm Tc Tc Em
8 × 108 2 × 106 8 × 106 1 × 108
θ, theta replication mechanism; Cm, chloramphenicol; Em, erythromycin; RC, rolling circle replication mechanism; Tc, tetracycline.
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harboring a chloramphenicol selection marker (pC194). High electroporation efficiency was also obtained with the larger plasmid carrying an erythromycin resistance gene (pMTL500Eres). The lowest transformation rates were obtained with plasmids containing tetracycline resistance marker. In this case, for the same plasmid size, the plasmid with a high copy number (pT181) gave better results than the plasmid with a low copy number (pLS1). Those results showed that chloramphenicol and erythromycin are more suitable selection markers for the electro-transformation of B. cereus. With the appropriate selection marker, the described electroporation protocol gave high transformation efficiencies, even for large theta replicating plasmids with low copy number per cell (Table 2). Interesting results were also obtained with two other strains of B. cereus (6 × 106 cfu μg− 1 ml− 1 with HER1399 and 3 × 106 cfu μg− 1 ml− 1 with HER-1414 with pC194 DNA). Those results are 103 times superior than previously reported for B. cereus strains. Therefore, this protocol can increase the electro-transformation efficiencies for several strains of B. cereus, but growth and electroporation parameters should be adapted to each specific strain, as previously reported for other bacterial species (Buckley et al., 1999). This paper describes an electro-transformation method for B. cereus ATCC14579 with results of 2 × 109 cfu μg− 1 ml− 1; this level of transformation is the highest ever reported for Bacillus sp. The method is quick, simple, highly reproducible, appropriate for many plasmids and suitable for many genetic manipulations that are not possible without high-efficiency transformation protocols. In addition, this procedure may be useful for other Bacillus strains that are refractory to electroporation. Acknowledgements We are very grateful to Gloria del Solar, MarieFrançoise Noirot-Gros, Sleem Khan and Laurent Jannière for the generous provision of the pLS1, pC194, pT181 and pMTL500Eres plasmids. We also acknowledge Felix d'Hérelle Reference Center for Bacterial Viruses for providing B. cereus strains HER1399 and HER-1414. We thank the members of the Groupe de Recherche en Santé Respiratoire for helpful discussions. NT is the recipient of a postdoctoral fellowship from the Groupe de Recherche en Santé Respiratoire. CD acknowledges a CIHR/IRSST scholarship. This work was funded by Defense Research and Development Canada Suffield.
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