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An improved method for the electrotransformation of lactic acid bacteria: A comparative survey
Journal of Microbiological Methods 105 (2014) 130–133
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
An improved method for the electrotransformation of lactic acid bacteria: A comparative survey José Mª. Landete ⁎, Juan L. Arqués, Ángela Peirotén, Susana Langa, Margarita Medina Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de La Coruña km 7.5, 28040 Madrid, Spain
a r t i c l e
i n f o
Article history: Received 21 July 2014 Accepted 21 July 2014 Available online 30 July 2014 Keywords: Lactic acid bacteria Transformation Lactobacillus reuteri Voltage
a b s t r a c t An efficient method for genetic transformation of lactic acid bacteria (LAB) by electroporation is presented in this work. A comparative survey with other electrotransformation methods already published showed that the method proposed here yields the higher electrotransformation efficiency in the 12 LAB strains tested, which could make the method applicable to other LAB species or genera. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Lactic acid bacteria (LAB) are Generally Recognized as Safe (GRAS) organisms involved in the manufacture of a wide variety of fermented foods. To develop genetic engineering techniques for LAB vector construction provides an alternative method for improving these microorganisms since it opens up the possibility of their modification by introducing new genes that give rise to desirable characteristics and novel phenotypes, by modifying their metabolic functions or removing unwanted traits (Shareck et al., 2004). To date, several protocols for the transformation of LAB have been developed. The aim of our work was to improve the electrotransformation method already developed for LAB, allowing the transformation of other LAB strains resistant to this technique. Moreover, the efficiency of the method proposed in this paper was successfully compared with other previously published methods. 2. Materials and methods 2.1. Bacterial strains and plasmid Lactobacillus, Enterococcus, Pediococcus, Leuconostoc and Streptococcus strains (Table 1) were routinely cultivated under anaerobic conditions at 37 °C in MRS broth (Scharlau Chemie SA, Barcelona, Spain). Lactococcus lactis strains were grown at 30 °C in M17 medium (Scharlau Chemie SA) supplemented with glucose (5 g/L) (GM17). The pT1NX vector (Schotte et al., 2000) was used for selection in LAB. A final concentration of 8 μg/mL erythromycin (Sigma Chemical ⁎ Corresponding author. E-mail address: [email protected] (J.M.ª Landete).
http://dx.doi.org/10.1016/j.mimet.2014.07.022 0167-7012/© 2014 Elsevier B.V. All rights reserved.
Co., St. Louis, MO) was used for the selection of transformed Lactobacillus strains and 5 μg/mL for the rest of LAB. 2.2. An improved method for electrotransformation of LAB For the preparation of competent LAB, an overnight culture was inoculated 1:50 in GM17 (Lactococcus strains) or MRS broth (the other LAB strains) containing 1% glycine and 0.5 M sucrose and incubated at 30 °C or 37 °C respectively until an OD600 of 0.6 was reached. Bacteria were collected by centrifugation at 10,000 g, for 10 min at 4 °C and the pellet was washed three times in a washing solution (5 mM KH2PO4, 2 mM MgCl2, 10% glycerol (v/v)) containing 0.5 M (for Lactococcus) or 0.3 M (for other LAB) sucrose. Bacteria were resuspended 1:100 in the same solution and a volume of 40 or 80 μL was electroporated immediately or kept at 70 °C for further use; 2 μL of pT1NX vector (0.4 ng/μL) was added to the 40 μL aliquot of the resuspended Lactococcus or to the 80 μL aliquot of the rest of the resuspended LAB. LAB strains were electroporated at 1.7 kV (2.5 kV for Lactococcus strains), 200 Ω and 25 μF in 0.2 cm cuvettes using a BioRad GenePulser (BioRad, Life Science Research Products, CA, USA). After electroporation, Lactococcus strains were resuspended in GM17 broth containing 0.5 M sucrose, 20 mM MgCl2 and 2 mM CaCl2 and incubated at 30 °C for 2 h, whereas the rest of the LAB strains were resuspended in MRS broth containing 0.3 M sucrose, 20 mM MgCl2 and 2 mM CaCl2 and incubated anaerobically at 37 °C for 2.5 h. Following the incubation, Lactococcus strains were plated on M17 containing 0.5 M sucrose supplemented with erythromycin (5 μL/mL), and the rest of LAB strains were plated on MRS containing 0.3 M sucrose supplemented with erythromycin (8 μL/mL). The plates were incubated at 30 °C (Lactococcus) or 37 °C (rest of LAB) for 2 or 3 days under anaerobic conditions.
Table 1 Number of transformants/μg DNA (pT1NX) after electroporation of different LAB with the methods tested. Results are expressed as averages ± standard deviations (SD). Chassy and Flickinger (1987)
Holo and Nes (1989)
Cruz-Rodz and Gilmore (1990)
Lactococcus lactis subsp. cremoris MG1363 Lactococcus lactis subsp. lactis INIA TAB26 Lactobacillus reuteri CECT925 Lactobacillus reuteri INIA P569 Lactobacillus casei BL23
5.1 0.6 2.2 0.7 0
105 ± 105 104 ± 103
4.6 · 107 ± 0.3· 107 2.3 · 106 ± 0.1 · 106 0
4.5 1.2 4.1 0.5 0
0
0
Lactobacillus brevis INIA ESI38 Lactobacillus rhamnosus INIA P426 Enterococcus faecium INIA TAB7 Enterococcus faecalis INIA P127 Pediococcus acidilactici PAC 1.0 Leuconostoc dextranicum CECT912 Streptococcus thermophilus St20
· · · ·
0 7.1 0.5 7.1 1.6 4.2 1.8 4.1 0.6 0
· · · · · · · ·
4
10 ± 104 102 ± 102 10 ± 10 104 ± 104
0 0
2.1 0.1 1.1 0.2 7.3 1.5 1.5 0.7 2.8 1.1 0
· · · · · · · · · ·
5
10 ± 105 103 ± 103 10 ± 10 106 ± 106 102 ± 102
0 3
4.3 · 10 ± 13 · 103
5
6.0 · 10 ± 1.2 · 105
· · · ·
106 ± 106 05 ± 105
Ahrné et al. (1992) 3.6 0.8 4.2 0.4 0
· · · ·
102 ± 102 102 ± 102
0
3.1 0.6 4.1 1.1 0
· · · ·
1.2 0.5 8.1 0.9 2.7 0.9 3.6 0.1 4.2 1.4
· · · · · · · · · ·
3
10 ± 103 102 ± 102
2
8.1 · 10 ± 0.7 · 102 0 0
106 106 102 102 104 104 102 102 106 104
±
2.4 · 102 ± 0.3 · 102 0
±
0
±
0
±
±
3
1.1 · 10 ± 0.4 · 103
Kim et al. (1992)
Varmanen et al (1998)
O'Sullivan and Fitzgerald (1999)
Serror et al. (2002)
Leathers et al. (2004)
1.2 · 106 ± 0–9 · 105 1.2 · 105 ± 0.8 · 104 0
1.2 · 107 ± 0.7 · 107 1.1 · 106 ± 0.7 · 105 10 ± 10.5
6.7 0.1 7.1 0.6 0
104 ± 104 104 ± 104
1.8 · 107 ± 0.8 · 106 1.6 · ·106 ± 0.6 · 105 5 ± 2.7
5.2 0.3 7.2 0.9 0
0
0
0
0
0
3.3 0.7 1.4 0.8 1.1 0.1 1.9 0.4 9.1 0.5 7.1 0.5 1.2 0.6 1.9 0.4
· · · · · · · · · · · · · · · ·
4
10 104 103 103 103 103 106 106 103 103 104 104 103 104 106 104
± ± ± ± ± ± ± ±
6.8 0.8 5.4 2.1 3.1 1.1 1.8 1.1 2.2 0.2 1.9 0.3 1.7 0.7 7.8 2.1
· · · · · · · · · · · · · · · ·
4
10 104 103 103 103 103 104 104 103 103 103 103 102 102 104 104
±
· · · ·
2
±
1.2 · 10 ± 0.4 · 102 0
±
0
± ±
7.7 · 102 ± 1.0 · 102 0
±
0
±
0
±
6.4 1.1 8.2 2.2 1.3 0.7 6.7 0.6 3.5 1.1 0
· · · · · · · · · ·
4
10 104 103 103 102 102 103 103 102 102
± ± ±
4.9 · 10 ± 0.2 · 104
· · · ·
104 ± 104 104 ± 104
4
10 ± 104 102 ± 102
±
7.1 · 103 ± 1.4 · 103 0
4.7 · 103 ± 2.0 · 103
1.3 · 103 ± 0.8 · 103 8.4 · 102 ± 0.8 · 102 8.8 · 103 ± 1.8 · 103
±
0 4
4.7 1.0 2.2 0.3 0
· · · ·
Present work
Source or reference
6.7 · 107 ± 0.4 · 107 7.1 · 106 ± 1.2 · 106 3.0 · 102 ± 0.2 · 102 1.1 · 10 ± 9.0 5.8 · 106 ± 1.4 · 104 5.1 · 103 ± 1.9 · 103 5.3 · 103 ± 0.6 · 103 4.5 · 107 ± 1.2 · 104 7.1 · 104 ± 1.3 · 104 1.6 · 105 ± 0.6 · 104 7.3 · 103 ± 1.4 · 103 8.1 · 107 ± 1.2 · 107
Gasson (1983) Arqués et al. (2005) CECT925 Rodríguez et al. (2003) Dr. Chassy, Univ. Illinois, USA Cogan et al. (1997) Rodríguez et al. (2012) Rodríguez et al. (2000) Rodríguez et al. (2012) Chikindas et al. (1993) CECT912 Dr. Cocconcelli, UCSC, Italy
J.M.ª Landete et al. / Journal of Microbiological Methods 105 (2014) 130–133
Strain
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J.M.ª Landete et al. / Journal of Microbiological Methods 105 (2014) 130–133
At least three independent replicates of each transformation were obtained. 2.3. Previous transformation procedures for LAB strains Different electrotransformation methods published (Table 2) for Lactobacillus casei (Chassy and Flickinger, 1987), Lactococcus lactis cremoris (Holo and Nes, 1989), Enterococcus faecalis (Cruz-Rodz and Gilmore, 1990), Lactobacillus reuteri (Ahrné et al., 1992), Pediococcus acidilactici (Kim et al., 1992), Lactobacillus rhamnosus (Varmanen et al., 1998), Streptococcus thermophilus (O'Sullivan and Fitzgerald, 1999), Lactobacillus delbrueckii (Serror et al., 2002) and Leuconostoc mesenteroides (Leathers et al., 2004) were assessed. At least three independent replicates of each transformation were obtained. 3. Results and discussion 3.1. An improved method for electrotransformation of LAB The modifications of the transformation method developed by Holo and Nes (1989) performed here made possible the transformation of different LAB species. Results for the different strains tested are shown in Table 1. The key points of the electrotransformation protocols were modified in order to obtain the enhanced procedure described here. First, MRS was chosen as growth media instead of GM17 for Lactobacillus, Enterococcus, Pediococcus, Leuconostoc and Streptococcus strains. With the purpose of increasing membrane permeability and enhancing the transformation rate, the LAB strains were grown in the presence of glycine and/or sucrose. The highest transformation efficiencies were obtained when cells were incubated with both components. However, the addition of raffinose or lysozyme did not increase the transformation efficiency in the strains tested. Glycine is the most common additive to compromise the cell wall and has been used to improve transformation efficiency in LAB (Holo and Nes, 1989; Hashiba et al., 1990; Kim et al., 1992; Gerber and Solioz, 2007). Lactococcus were washed with 0.5 M sucrose and 10% glycerol as previously described by Holo and Nes (1989). However, the rest of the
LAB strains were washed with 0.3 M sucrose and 10% glycerol. The addition of 5 mM KH2PO4 pH 7.4 and 2 mM MgCl2 in the washed solution, as proposed by Leathers et al. (2004), increased the transformation efficiency. Osmotic stabilization of cells with 0.5 M sucrose was also found to be critical during glycine treatment similar to other works (Cruz-Rodz and Gilmore, 1990). Moreover, in this study, we also improved the transformation efficiency by using a buffer containing KH2PO4 and MgCl2. Storage of competent strains at −70 °C in 0.5 M (for Lactococcus) or 0.3 M (for other LAB) sucrose containing 10% glycerol did not affect their transformability. Thus, competent cells can be prepared routinely in 40 or 80 μL and kept at −70 °C until use. High cell densities and high voltages in the presence of 10% glycerol caused an increase in time constant and reduced the risk of arcing. These frozen cells, when thawed in an ice-water bath and pulsed immediately, yielded the same (or higher) transformation frequencies as cells from the same batch before freezing. Consequently, glycerol was added to the cell suspensions before electroporation and, for simplicity, it was included in the medium used for freezing the cells. The optimized volume was 40 μL for Lactococcus and 80 μL for the rest of the LAB strains tested. An increase or reduction of that optimized volume led to a decrease in the transformation efficiencies. Optimizing the voltage was considered a crucial factor for obtaining the successful transformation of all the LAB strains tested. The transformation efficiencies increased when the voltage was enhanced to 2.5 kV for Lactococcus strains, and to 1.7 kV for the rest of microorganisms assayed. Voltage higher and lower than 1.7 kV produced a decrease in the transformation efficiencies of Lactobacillus, Pediococcus and Leuconostoc. Resistance between 100 Ω and 800 Ω were also tested, observing that at 200 Ω the transformation frequencies were higher for these voltage values. 3.2. A comparative survey on different electrotransformation methods A comparison of the transformation efficiencies between our protocol and other nine published methods which were carried out with 12 different LAB strains is shown in Table 1. The electrotransformation protocol proposed here was the most versatile since it allowed the
Table 2 Protocols used to transform LAB in this work. LAB
Grown
Washed
Voltage Resistance Cultured
Reference
L. casei
MRS
1.0 kV
100
LCM, glucose 1 g/L
L. lactis
GM17, 1% glycine, 0.5 M sucrose
7 mM HEPES pH 7.4, 272 mM sucrose, 1 mM MgCl2 0.5 M sucrose, 10% glycerol
2.0 kV
200
E. faecalis
GM17, 0.5 M sucrose, 8% glycine
0.5 M sucrose, 10% glycerol
2.5 kV
200
L. reuteri
LCM-G
1 mM HEPES pH 7, 0.5 M raffinose
2.5 kV
200
GM17, 0.5 M sucrose, 20 mM MgCl2, 2 mM CaCl2 GM17, 0.5 M sucrose, 20 mM MgCl2, 2 mM CaCl2 LCM-G
Chassy and Flickinger (1987) Holo and Nes (1989)
P. acidilactici
TGE, 20 mM threonine
2.5 kV
200
TGE 0.5 M sucrose
L. rhamnosus
MRS, 2% glycine
1.5 kV
200
S. thermophilus
2.5 kV
200
MRS, 20 mM MgCl2, 2 mM Varmanen et al. CaCl2 (1998) Belliker broth O'Sullivan and Fitzgerald (1999)
L. delbrueckii
Belliker broth, 20 mM D-L threonine MRS, 0.1% glycine
7 mM potassium phosphate, 0.6 M sucrose, lysozyme 4000 U/mL, 1 mM MgCl2 7 mM potassium phosphate pH 7.4, 0.5 M sucrose, 1 mM MgCl2 HEPES, 1 mM MgCl2
1.0 kV
800
MRS
L. mesenteroides
MRS-V8
2.0 kV
400
MRS-V8
Lactococcus
GM17, 1% glycine, 0.5 M sucrose
0.4 M sucrose, 5 mM KH2PO4, 1 mM MgCl2. 45 °C 20 min 1 mM K2HPO4/KH2PO4 pH 7.4, 1 mM MgCl2, 0.5 M sucrose 0.5 M sucrose, 10% glycerol, 5 mM KH2PO4 pH 7.4, 2 mM MgCl2
2.5 kV
200
Lactobacillus, Pediococcus, Enterococcus Leuconostoc and Streptococcus
MRS, 1% glycine, 0.3 M sucrose
0.3 M sucrose, 10% glycerol, 5 mM KH2PO4 pH 7.4, 2 mM MgCl2
1.7 kV
200
GM17, 0.5 M sucrose, 20 mM MgCl2, 2 mM CaCl2 MRS, 0.3 M sucrose, 20 mM MgCl2, 2 mM CaCl2
Cruz-Rodz and Gilmore (1990) Ahrné et al. (1992) Kim et al. (1992)
Serror et al. (2002) Leathers et al. (2004) Present work
Present work
J.M.ª Landete et al. / Journal of Microbiological Methods 105 (2014) 130–133
transformation of all the strains tested. However, the methods proposed by Varmanen et al. (1998) and by Kim et al. (1992) failed in transforming one or two of the L. reuteri strains tested, respectively. The method proposed by Cruz-Rodz and Gilmore (1990), also based on Holo and Nes (1989), was able to transform different species of LAB, with the exception of L. reuteri and L. rhamnosus. All methods tested allowed the transformation of L. lactis subsp. cremoris MG1363, L. lactis subsp. lactis INIA TAB26, L. casei BL23, Enterococcus faecium INIA TAB7 and S. thermophilus St20, although the transformation efficiencies varied between the different methods. Our protocol reached the highest transformation efficiencies and L. lactis subsp. cremoris MG1363 was the strain most efficiently transformed by the different protocols studied. As a conclusion, the improved electrotransformation method proposed in this work allowed the transformation of all the LAB strains tested, and it is likely to be applicable to other LAB or even other Gram-positive organisms. Moreover, transformation efficiencies were increased with this method, allowing the transformation of strains previously resistant as L. reuteri. Acknowledgments This work was supported by projects RTA2010-00116-00-00 and RM2012-00004-00-00. J.M. Landete has a postdoctoral contract with the research program “Ramón y Cajal” (MINECO, Spain). References Ahrné, S.,Molin, G.,Axelsson, L., 1992. Transformation of Lactobacillus reuteri with electroporation: studies on the erythromycin resistance plasmid pLUL631. Curr. Microbiol. 24, 199–205. Arqués, J.L.,Rodríguez, E.,Gaya, P.,Medina, M.,Guamis, M.,Nuñez, M., 2005. Inactivation of Staphylococcus aureus in raw milk cheese by combinations of high-pressure treatments and bacteriocin-producing lactic acid bacteria. J. Appl. Microbiol. 98, 254–260. Chassy, B.M.,Flickinger, J.L., 1987. Transformation of Lactobacillus casei by electroporation. FEMS Microbiol. Lett. 44, 173–177. Chikindas, M.L.,García-Garcerá, M.J.,Driessen, A.J.,Ledeboer, A.M.,Nissen-Meyer, J.,Nes, I.F., Abee, T., Konings, W.N., Venema, G., 1993. Pediocin PA-1, a bacteriocin from
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Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells. Appl. Environ. Microbiol. 59, 3577–3584. Cogan, T.M., Barbosa, M., Beuvier, E., Bianchi-Salvadori, B., Cocconcelli, P.S., Fernandes, I., Gomez, J., Kalantzopoulos, G., Ledda, A., Medina, M., Rea, M.C., Rodríguez, E., 1997. Characterization of the lactic acid bacteria in artisanal dairy products. J. Dairy Res. 64, 409–421. Cruz-Rodz, A.L.,Gilmore, M.S., 1990. High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation. Mol. Gen. Genet. 224, 152–154. Gasson, M.J., 1983. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J. Bacteriol. 154, 1–9. Gerber, S., Solioz, M., 2007. Efficient transformation of Lactococcus lactis IL1403 and generation of knock-out mutants by homologous recombination. J. Basic Microbiol. 47, 281–286. Hashiba, H., Takiguchi, R., Iskii, S., Aoyama, K., 1990. Transformation of Lactobacillus helveticus ssp. jugurti with plasmid pLHR by electroporation. Agric. Biol. Chem. 54, 1537–1541. Holo, H.,Nes, I.F., 1989. High-frequency transformation, by electroporation of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl. Environ. Microbiol. 55, 3119–3123. Kim, W.J., Ray, B., Johnson, M.C., 1992. Plasmid transfers by conjugation and electroporation in Pediococcus acidilactici. J. Appl. Bacteriol. 72, 201–207. Leathers, T.D., Jones, J.D., Wyckoff, H.A., 2004. Transformation of alternan-producing strains of Leuconostoc by electroporation. Biotechnol. Lett. 26, 1119–1124. O'Sullivan, T.F., Fitzgerald, G.F., 1999. Electrotransformation of industrial strains of Streptococcus thermophilus. J. Appl. Microbiol. 86, 275–283. Rodríguez, E., González, B., Gaya, P., Nuñez, M., Medina, M., 2000. Diversity of bacteriocins produced by lactic acid bacteria isolated from raw milk. Int. Dairy J. 10, 7–15. Rodríguez, E., Arqués, J.L., Rodríguez, R., Nuñez, M., Medina, M., 2003. Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits. Lett. Appl. Microbiol. 37, 259–263. Rodríguez, E., Arqués, J.L., Rodríguez, R., Landete, J.M., Medina, M., 2012. Antimicrobial properties of probiotic strains isolated from breast-fed infants. J. Funct. Foods 4, 542–551. Schotte, L.,Steidler, L.,Vandekerckhove, J.,Remaut, E., 2000. Secretion of biologically active murine interleukin-10 by Lactococcus lactis. Enzym. Microb. Technol. 27, 761–765. Serror, P., Sasaki, T., Ehrlich, D., Maguin, E., 2002. Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids. Appl. Environ. Microbiol. 68, 46–52. Shareck, J., Choi, Y., Lee, B., Miguez, C.B., 2004. Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology. Crit. Rev. Biotechnol. 24, 155–208. Varmanen, P.,Rantanen, T.,Palva, A.,Tynkkynen, S., 1998. Cloning and characterization of a prolinase gene (pepR) from Lactobacillus rhamnosus. Appl. Environ. Microbiol. 64, 1831–1836.
Contents lists available at ScienceDirect
Journal of Microbiological Methods journal homepage: www.elsevier.com/locate/jmicmeth
An improved method for the electrotransformation of lactic acid bacteria: A comparative survey José Mª. Landete ⁎, Juan L. Arqués, Ángela Peirotén, Susana Langa, Margarita Medina Departamento de Tecnología de Alimentos, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (INIA), Carretera de La Coruña km 7.5, 28040 Madrid, Spain
a r t i c l e
i n f o
Article history: Received 21 July 2014 Accepted 21 July 2014 Available online 30 July 2014 Keywords: Lactic acid bacteria Transformation Lactobacillus reuteri Voltage
a b s t r a c t An efficient method for genetic transformation of lactic acid bacteria (LAB) by electroporation is presented in this work. A comparative survey with other electrotransformation methods already published showed that the method proposed here yields the higher electrotransformation efficiency in the 12 LAB strains tested, which could make the method applicable to other LAB species or genera. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Lactic acid bacteria (LAB) are Generally Recognized as Safe (GRAS) organisms involved in the manufacture of a wide variety of fermented foods. To develop genetic engineering techniques for LAB vector construction provides an alternative method for improving these microorganisms since it opens up the possibility of their modification by introducing new genes that give rise to desirable characteristics and novel phenotypes, by modifying their metabolic functions or removing unwanted traits (Shareck et al., 2004). To date, several protocols for the transformation of LAB have been developed. The aim of our work was to improve the electrotransformation method already developed for LAB, allowing the transformation of other LAB strains resistant to this technique. Moreover, the efficiency of the method proposed in this paper was successfully compared with other previously published methods. 2. Materials and methods 2.1. Bacterial strains and plasmid Lactobacillus, Enterococcus, Pediococcus, Leuconostoc and Streptococcus strains (Table 1) were routinely cultivated under anaerobic conditions at 37 °C in MRS broth (Scharlau Chemie SA, Barcelona, Spain). Lactococcus lactis strains were grown at 30 °C in M17 medium (Scharlau Chemie SA) supplemented with glucose (5 g/L) (GM17). The pT1NX vector (Schotte et al., 2000) was used for selection in LAB. A final concentration of 8 μg/mL erythromycin (Sigma Chemical ⁎ Corresponding author. E-mail address: [email protected] (J.M.ª Landete).
http://dx.doi.org/10.1016/j.mimet.2014.07.022 0167-7012/© 2014 Elsevier B.V. All rights reserved.
Co., St. Louis, MO) was used for the selection of transformed Lactobacillus strains and 5 μg/mL for the rest of LAB. 2.2. An improved method for electrotransformation of LAB For the preparation of competent LAB, an overnight culture was inoculated 1:50 in GM17 (Lactococcus strains) or MRS broth (the other LAB strains) containing 1% glycine and 0.5 M sucrose and incubated at 30 °C or 37 °C respectively until an OD600 of 0.6 was reached. Bacteria were collected by centrifugation at 10,000 g, for 10 min at 4 °C and the pellet was washed three times in a washing solution (5 mM KH2PO4, 2 mM MgCl2, 10% glycerol (v/v)) containing 0.5 M (for Lactococcus) or 0.3 M (for other LAB) sucrose. Bacteria were resuspended 1:100 in the same solution and a volume of 40 or 80 μL was electroporated immediately or kept at 70 °C for further use; 2 μL of pT1NX vector (0.4 ng/μL) was added to the 40 μL aliquot of the resuspended Lactococcus or to the 80 μL aliquot of the rest of the resuspended LAB. LAB strains were electroporated at 1.7 kV (2.5 kV for Lactococcus strains), 200 Ω and 25 μF in 0.2 cm cuvettes using a BioRad GenePulser (BioRad, Life Science Research Products, CA, USA). After electroporation, Lactococcus strains were resuspended in GM17 broth containing 0.5 M sucrose, 20 mM MgCl2 and 2 mM CaCl2 and incubated at 30 °C for 2 h, whereas the rest of the LAB strains were resuspended in MRS broth containing 0.3 M sucrose, 20 mM MgCl2 and 2 mM CaCl2 and incubated anaerobically at 37 °C for 2.5 h. Following the incubation, Lactococcus strains were plated on M17 containing 0.5 M sucrose supplemented with erythromycin (5 μL/mL), and the rest of LAB strains were plated on MRS containing 0.3 M sucrose supplemented with erythromycin (8 μL/mL). The plates were incubated at 30 °C (Lactococcus) or 37 °C (rest of LAB) for 2 or 3 days under anaerobic conditions.
Table 1 Number of transformants/μg DNA (pT1NX) after electroporation of different LAB with the methods tested. Results are expressed as averages ± standard deviations (SD). Chassy and Flickinger (1987)
Holo and Nes (1989)
Cruz-Rodz and Gilmore (1990)
Lactococcus lactis subsp. cremoris MG1363 Lactococcus lactis subsp. lactis INIA TAB26 Lactobacillus reuteri CECT925 Lactobacillus reuteri INIA P569 Lactobacillus casei BL23
5.1 0.6 2.2 0.7 0
105 ± 105 104 ± 103
4.6 · 107 ± 0.3· 107 2.3 · 106 ± 0.1 · 106 0
4.5 1.2 4.1 0.5 0
0
0
Lactobacillus brevis INIA ESI38 Lactobacillus rhamnosus INIA P426 Enterococcus faecium INIA TAB7 Enterococcus faecalis INIA P127 Pediococcus acidilactici PAC 1.0 Leuconostoc dextranicum CECT912 Streptococcus thermophilus St20
· · · ·
0 7.1 0.5 7.1 1.6 4.2 1.8 4.1 0.6 0
· · · · · · · ·
4
10 ± 104 102 ± 102 10 ± 10 104 ± 104
0 0
2.1 0.1 1.1 0.2 7.3 1.5 1.5 0.7 2.8 1.1 0
· · · · · · · · · ·
5
10 ± 105 103 ± 103 10 ± 10 106 ± 106 102 ± 102
0 3
4.3 · 10 ± 13 · 103
5
6.0 · 10 ± 1.2 · 105
· · · ·
106 ± 106 05 ± 105
Ahrné et al. (1992) 3.6 0.8 4.2 0.4 0
· · · ·
102 ± 102 102 ± 102
0
3.1 0.6 4.1 1.1 0
· · · ·
1.2 0.5 8.1 0.9 2.7 0.9 3.6 0.1 4.2 1.4
· · · · · · · · · ·
3
10 ± 103 102 ± 102
2
8.1 · 10 ± 0.7 · 102 0 0
106 106 102 102 104 104 102 102 106 104
±
2.4 · 102 ± 0.3 · 102 0
±
0
±
0
±
±
3
1.1 · 10 ± 0.4 · 103
Kim et al. (1992)
Varmanen et al (1998)
O'Sullivan and Fitzgerald (1999)
Serror et al. (2002)
Leathers et al. (2004)
1.2 · 106 ± 0–9 · 105 1.2 · 105 ± 0.8 · 104 0
1.2 · 107 ± 0.7 · 107 1.1 · 106 ± 0.7 · 105 10 ± 10.5
6.7 0.1 7.1 0.6 0
104 ± 104 104 ± 104
1.8 · 107 ± 0.8 · 106 1.6 · ·106 ± 0.6 · 105 5 ± 2.7
5.2 0.3 7.2 0.9 0
0
0
0
0
0
3.3 0.7 1.4 0.8 1.1 0.1 1.9 0.4 9.1 0.5 7.1 0.5 1.2 0.6 1.9 0.4
· · · · · · · · · · · · · · · ·
4
10 104 103 103 103 103 106 106 103 103 104 104 103 104 106 104
± ± ± ± ± ± ± ±
6.8 0.8 5.4 2.1 3.1 1.1 1.8 1.1 2.2 0.2 1.9 0.3 1.7 0.7 7.8 2.1
· · · · · · · · · · · · · · · ·
4
10 104 103 103 103 103 104 104 103 103 103 103 102 102 104 104
±
· · · ·
2
±
1.2 · 10 ± 0.4 · 102 0
±
0
± ±
7.7 · 102 ± 1.0 · 102 0
±
0
±
0
±
6.4 1.1 8.2 2.2 1.3 0.7 6.7 0.6 3.5 1.1 0
· · · · · · · · · ·
4
10 104 103 103 102 102 103 103 102 102
± ± ±
4.9 · 10 ± 0.2 · 104
· · · ·
104 ± 104 104 ± 104
4
10 ± 104 102 ± 102
±
7.1 · 103 ± 1.4 · 103 0
4.7 · 103 ± 2.0 · 103
1.3 · 103 ± 0.8 · 103 8.4 · 102 ± 0.8 · 102 8.8 · 103 ± 1.8 · 103
±
0 4
4.7 1.0 2.2 0.3 0
· · · ·
Present work
Source or reference
6.7 · 107 ± 0.4 · 107 7.1 · 106 ± 1.2 · 106 3.0 · 102 ± 0.2 · 102 1.1 · 10 ± 9.0 5.8 · 106 ± 1.4 · 104 5.1 · 103 ± 1.9 · 103 5.3 · 103 ± 0.6 · 103 4.5 · 107 ± 1.2 · 104 7.1 · 104 ± 1.3 · 104 1.6 · 105 ± 0.6 · 104 7.3 · 103 ± 1.4 · 103 8.1 · 107 ± 1.2 · 107
Gasson (1983) Arqués et al. (2005) CECT925 Rodríguez et al. (2003) Dr. Chassy, Univ. Illinois, USA Cogan et al. (1997) Rodríguez et al. (2012) Rodríguez et al. (2000) Rodríguez et al. (2012) Chikindas et al. (1993) CECT912 Dr. Cocconcelli, UCSC, Italy
J.M.ª Landete et al. / Journal of Microbiological Methods 105 (2014) 130–133
Strain
131
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J.M.ª Landete et al. / Journal of Microbiological Methods 105 (2014) 130–133
At least three independent replicates of each transformation were obtained. 2.3. Previous transformation procedures for LAB strains Different electrotransformation methods published (Table 2) for Lactobacillus casei (Chassy and Flickinger, 1987), Lactococcus lactis cremoris (Holo and Nes, 1989), Enterococcus faecalis (Cruz-Rodz and Gilmore, 1990), Lactobacillus reuteri (Ahrné et al., 1992), Pediococcus acidilactici (Kim et al., 1992), Lactobacillus rhamnosus (Varmanen et al., 1998), Streptococcus thermophilus (O'Sullivan and Fitzgerald, 1999), Lactobacillus delbrueckii (Serror et al., 2002) and Leuconostoc mesenteroides (Leathers et al., 2004) were assessed. At least three independent replicates of each transformation were obtained. 3. Results and discussion 3.1. An improved method for electrotransformation of LAB The modifications of the transformation method developed by Holo and Nes (1989) performed here made possible the transformation of different LAB species. Results for the different strains tested are shown in Table 1. The key points of the electrotransformation protocols were modified in order to obtain the enhanced procedure described here. First, MRS was chosen as growth media instead of GM17 for Lactobacillus, Enterococcus, Pediococcus, Leuconostoc and Streptococcus strains. With the purpose of increasing membrane permeability and enhancing the transformation rate, the LAB strains were grown in the presence of glycine and/or sucrose. The highest transformation efficiencies were obtained when cells were incubated with both components. However, the addition of raffinose or lysozyme did not increase the transformation efficiency in the strains tested. Glycine is the most common additive to compromise the cell wall and has been used to improve transformation efficiency in LAB (Holo and Nes, 1989; Hashiba et al., 1990; Kim et al., 1992; Gerber and Solioz, 2007). Lactococcus were washed with 0.5 M sucrose and 10% glycerol as previously described by Holo and Nes (1989). However, the rest of the
LAB strains were washed with 0.3 M sucrose and 10% glycerol. The addition of 5 mM KH2PO4 pH 7.4 and 2 mM MgCl2 in the washed solution, as proposed by Leathers et al. (2004), increased the transformation efficiency. Osmotic stabilization of cells with 0.5 M sucrose was also found to be critical during glycine treatment similar to other works (Cruz-Rodz and Gilmore, 1990). Moreover, in this study, we also improved the transformation efficiency by using a buffer containing KH2PO4 and MgCl2. Storage of competent strains at −70 °C in 0.5 M (for Lactococcus) or 0.3 M (for other LAB) sucrose containing 10% glycerol did not affect their transformability. Thus, competent cells can be prepared routinely in 40 or 80 μL and kept at −70 °C until use. High cell densities and high voltages in the presence of 10% glycerol caused an increase in time constant and reduced the risk of arcing. These frozen cells, when thawed in an ice-water bath and pulsed immediately, yielded the same (or higher) transformation frequencies as cells from the same batch before freezing. Consequently, glycerol was added to the cell suspensions before electroporation and, for simplicity, it was included in the medium used for freezing the cells. The optimized volume was 40 μL for Lactococcus and 80 μL for the rest of the LAB strains tested. An increase or reduction of that optimized volume led to a decrease in the transformation efficiencies. Optimizing the voltage was considered a crucial factor for obtaining the successful transformation of all the LAB strains tested. The transformation efficiencies increased when the voltage was enhanced to 2.5 kV for Lactococcus strains, and to 1.7 kV for the rest of microorganisms assayed. Voltage higher and lower than 1.7 kV produced a decrease in the transformation efficiencies of Lactobacillus, Pediococcus and Leuconostoc. Resistance between 100 Ω and 800 Ω were also tested, observing that at 200 Ω the transformation frequencies were higher for these voltage values. 3.2. A comparative survey on different electrotransformation methods A comparison of the transformation efficiencies between our protocol and other nine published methods which were carried out with 12 different LAB strains is shown in Table 1. The electrotransformation protocol proposed here was the most versatile since it allowed the
Table 2 Protocols used to transform LAB in this work. LAB
Grown
Washed
Voltage Resistance Cultured
Reference
L. casei
MRS
1.0 kV
100
LCM, glucose 1 g/L
L. lactis
GM17, 1% glycine, 0.5 M sucrose
7 mM HEPES pH 7.4, 272 mM sucrose, 1 mM MgCl2 0.5 M sucrose, 10% glycerol
2.0 kV
200
E. faecalis
GM17, 0.5 M sucrose, 8% glycine
0.5 M sucrose, 10% glycerol
2.5 kV
200
L. reuteri
LCM-G
1 mM HEPES pH 7, 0.5 M raffinose
2.5 kV
200
GM17, 0.5 M sucrose, 20 mM MgCl2, 2 mM CaCl2 GM17, 0.5 M sucrose, 20 mM MgCl2, 2 mM CaCl2 LCM-G
Chassy and Flickinger (1987) Holo and Nes (1989)
P. acidilactici
TGE, 20 mM threonine
2.5 kV
200
TGE 0.5 M sucrose
L. rhamnosus
MRS, 2% glycine
1.5 kV
200
S. thermophilus
2.5 kV
200
MRS, 20 mM MgCl2, 2 mM Varmanen et al. CaCl2 (1998) Belliker broth O'Sullivan and Fitzgerald (1999)
L. delbrueckii
Belliker broth, 20 mM D-L threonine MRS, 0.1% glycine
7 mM potassium phosphate, 0.6 M sucrose, lysozyme 4000 U/mL, 1 mM MgCl2 7 mM potassium phosphate pH 7.4, 0.5 M sucrose, 1 mM MgCl2 HEPES, 1 mM MgCl2
1.0 kV
800
MRS
L. mesenteroides
MRS-V8
2.0 kV
400
MRS-V8
Lactococcus
GM17, 1% glycine, 0.5 M sucrose
0.4 M sucrose, 5 mM KH2PO4, 1 mM MgCl2. 45 °C 20 min 1 mM K2HPO4/KH2PO4 pH 7.4, 1 mM MgCl2, 0.5 M sucrose 0.5 M sucrose, 10% glycerol, 5 mM KH2PO4 pH 7.4, 2 mM MgCl2
2.5 kV
200
Lactobacillus, Pediococcus, Enterococcus Leuconostoc and Streptococcus
MRS, 1% glycine, 0.3 M sucrose
0.3 M sucrose, 10% glycerol, 5 mM KH2PO4 pH 7.4, 2 mM MgCl2
1.7 kV
200
GM17, 0.5 M sucrose, 20 mM MgCl2, 2 mM CaCl2 MRS, 0.3 M sucrose, 20 mM MgCl2, 2 mM CaCl2
Cruz-Rodz and Gilmore (1990) Ahrné et al. (1992) Kim et al. (1992)
Serror et al. (2002) Leathers et al. (2004) Present work
Present work
J.M.ª Landete et al. / Journal of Microbiological Methods 105 (2014) 130–133
transformation of all the strains tested. However, the methods proposed by Varmanen et al. (1998) and by Kim et al. (1992) failed in transforming one or two of the L. reuteri strains tested, respectively. The method proposed by Cruz-Rodz and Gilmore (1990), also based on Holo and Nes (1989), was able to transform different species of LAB, with the exception of L. reuteri and L. rhamnosus. All methods tested allowed the transformation of L. lactis subsp. cremoris MG1363, L. lactis subsp. lactis INIA TAB26, L. casei BL23, Enterococcus faecium INIA TAB7 and S. thermophilus St20, although the transformation efficiencies varied between the different methods. Our protocol reached the highest transformation efficiencies and L. lactis subsp. cremoris MG1363 was the strain most efficiently transformed by the different protocols studied. As a conclusion, the improved electrotransformation method proposed in this work allowed the transformation of all the LAB strains tested, and it is likely to be applicable to other LAB or even other Gram-positive organisms. Moreover, transformation efficiencies were increased with this method, allowing the transformation of strains previously resistant as L. reuteri. Acknowledgments This work was supported by projects RTA2010-00116-00-00 and RM2012-00004-00-00. J.M. Landete has a postdoctoral contract with the research program “Ramón y Cajal” (MINECO, Spain). References Ahrné, S.,Molin, G.,Axelsson, L., 1992. Transformation of Lactobacillus reuteri with electroporation: studies on the erythromycin resistance plasmid pLUL631. Curr. Microbiol. 24, 199–205. Arqués, J.L.,Rodríguez, E.,Gaya, P.,Medina, M.,Guamis, M.,Nuñez, M., 2005. Inactivation of Staphylococcus aureus in raw milk cheese by combinations of high-pressure treatments and bacteriocin-producing lactic acid bacteria. J. Appl. Microbiol. 98, 254–260. Chassy, B.M.,Flickinger, J.L., 1987. Transformation of Lactobacillus casei by electroporation. FEMS Microbiol. Lett. 44, 173–177. Chikindas, M.L.,García-Garcerá, M.J.,Driessen, A.J.,Ledeboer, A.M.,Nissen-Meyer, J.,Nes, I.F., Abee, T., Konings, W.N., Venema, G., 1993. Pediocin PA-1, a bacteriocin from
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Pediococcus acidilactici PAC1.0, forms hydrophilic pores in the cytoplasmic membrane of target cells. Appl. Environ. Microbiol. 59, 3577–3584. Cogan, T.M., Barbosa, M., Beuvier, E., Bianchi-Salvadori, B., Cocconcelli, P.S., Fernandes, I., Gomez, J., Kalantzopoulos, G., Ledda, A., Medina, M., Rea, M.C., Rodríguez, E., 1997. Characterization of the lactic acid bacteria in artisanal dairy products. J. Dairy Res. 64, 409–421. Cruz-Rodz, A.L.,Gilmore, M.S., 1990. High efficiency introduction of plasmid DNA into glycine treated Enterococcus faecalis by electroporation. Mol. Gen. Genet. 224, 152–154. Gasson, M.J., 1983. Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing. J. Bacteriol. 154, 1–9. Gerber, S., Solioz, M., 2007. Efficient transformation of Lactococcus lactis IL1403 and generation of knock-out mutants by homologous recombination. J. Basic Microbiol. 47, 281–286. Hashiba, H., Takiguchi, R., Iskii, S., Aoyama, K., 1990. Transformation of Lactobacillus helveticus ssp. jugurti with plasmid pLHR by electroporation. Agric. Biol. Chem. 54, 1537–1541. Holo, H.,Nes, I.F., 1989. High-frequency transformation, by electroporation of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl. Environ. Microbiol. 55, 3119–3123. Kim, W.J., Ray, B., Johnson, M.C., 1992. Plasmid transfers by conjugation and electroporation in Pediococcus acidilactici. J. Appl. Bacteriol. 72, 201–207. Leathers, T.D., Jones, J.D., Wyckoff, H.A., 2004. Transformation of alternan-producing strains of Leuconostoc by electroporation. Biotechnol. Lett. 26, 1119–1124. O'Sullivan, T.F., Fitzgerald, G.F., 1999. Electrotransformation of industrial strains of Streptococcus thermophilus. J. Appl. Microbiol. 86, 275–283. Rodríguez, E., González, B., Gaya, P., Nuñez, M., Medina, M., 2000. Diversity of bacteriocins produced by lactic acid bacteria isolated from raw milk. Int. Dairy J. 10, 7–15. Rodríguez, E., Arqués, J.L., Rodríguez, R., Nuñez, M., Medina, M., 2003. Reuterin production by lactobacilli isolated from pig faeces and evaluation of probiotic traits. Lett. Appl. Microbiol. 37, 259–263. Rodríguez, E., Arqués, J.L., Rodríguez, R., Landete, J.M., Medina, M., 2012. Antimicrobial properties of probiotic strains isolated from breast-fed infants. J. Funct. Foods 4, 542–551. Schotte, L.,Steidler, L.,Vandekerckhove, J.,Remaut, E., 2000. Secretion of biologically active murine interleukin-10 by Lactococcus lactis. Enzym. Microb. Technol. 27, 761–765. Serror, P., Sasaki, T., Ehrlich, D., Maguin, E., 2002. Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids. Appl. Environ. Microbiol. 68, 46–52. Shareck, J., Choi, Y., Lee, B., Miguez, C.B., 2004. Cloning vectors based on cryptic plasmids isolated from lactic acid bacteria: their characteristics and potential applications in biotechnology. Crit. Rev. Biotechnol. 24, 155–208. Varmanen, P.,Rantanen, T.,Palva, A.,Tynkkynen, S., 1998. Cloning and characterization of a prolinase gene (pepR) from Lactobacillus rhamnosus. Appl. Environ. Microbiol. 64, 1831–1836.