FEMS Microbiology Letters, 362, 2015, 1–6 doi: 10.1093/femsle/fnu033 Advance Access Publication Date: 4 December 2014 Research Letter
R E S E A R C H L E T T E R – Physiology & Biochemistry
High efficiency electrotransformation of Lactobacillus casei Dennis L. Welker1,∗ , Joanne E. Hughes1 , James L. Steele2 and Jeff R. Broadbent3 1
Department of Biology, Utah State University, Logan, UT 84322-5305, USA, 2 Department of Food Science, University of Wisconsin, Madison, WI 53706, USA and 3 Department of Nutrition, Dietetics and Food Sciences, Utah State University, Logan, UT 84322-8700, USA ∗ Corresponding author: Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322-5305, USA. Tel.: 435-797-3552; Fax: 435-797-1575; E-mail: [email protected] One sentence summary: This work describes a combined approach for raising the transformation efficiency of this species that may be of use for genetically modifying other Lactobacilli. Editor: Rustam Aminov
ABSTRACT We investigated whether protocols allowing high efficiency electrotransformation of other lactic acid bacteria were applicable to five strains of Lactobacillus casei (12A, 32G, A2-362, ATCC 334 and BL23). Addition of 1% glycine or 0.9 M NaCl during cell growth, limitation of the growth of the cell cultures to OD600 0.6–0.8, pre-electroporation treatment of cells with water or with a lithium acetate (100 mM)/dithiothreitol (10 mM) solution and optimization of electroporation conditions all improved transformation efficiencies. However, the five strains varied in their responses to these treatments. Transformation efficiencies of 106 colony forming units μg−1 pTRKH2 DNA and higher were obtained with three strains which is sufficient for construction of chromosomal gene knock-outs and gene replacements. Key words: electroporation; genetic manipulation; lactic acid bacteria; transformation
INTRODUCTION Lactobacillus casei and other lactic acid bacteria (LAB) are used in numerous food, agricultural and industrial fermentations. The ability to genetically manipulate L. casei and other LAB extends the range of studies that can be undertaken to understand their physiological and biochemical properties and allows for improvement of industrial strains (Kondo and Johansen 2002). However, construction of recombinant strains is often limited by the inability to introduce DNA constructs or to generate knockout mutations and gene replacements. The introduction of recombinant DNA by electrotransformation has been widely applied for genetic manipulation of LAB.
Electroporation of L. casei was first reported over 25 years ago (Chassy and Flickinger 1987) with subsequent reports describing the application of techniques, such as growth of the cells with glycine, to improve transformation efficiency (Luchansky, Muriana and Klaenhammer 1988; Natori, Kano and Imamoto 1990; Wei et al., 1995; Mason, Collins and Thompson 2005). The best transformation efficiencies reported for L. casei with thetareplicating plasmid vectors reached 105 to 106 colony forming units (CFU) μg−1 transforming DNA (Palomino et al., 2010), compared to typical efficiencies of 102 –104 CFU μg−1 transforming DNA. Those experiments used BL23 cells grown anaerobically in Lactobacillus MRS medium supplemented with 0.9 M NaCl,
Received: 26 September 2014; Accepted: 12 November 2014 C FEMS 2014. All rights reserved. For permissions, please e-mail: [email protected]
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which has been shown to reduce the amount of cell wall lipoteichoic acid, its mean chain length and its D-alanyl substitution (Palomino et al., 2013). Another new protocol involving treatment of cells with lithium acetate and dithiothreitol (DTT) was shown to increase the electrotransformation efficiency of Lactococcus lactis spp. lactis (Papagianni, Avramidis and Filioussis 2007; Spath, Heinl and Grabherr 2012), as well as L. plantarum and L. buchneri (Spath et al., 2012). Transformation efficiencies after lithium acetate/DTT treatment ranged from 105 to 109 CFU μg−1 transforming DNA for L. lactis (Papagianni et al., 2007; Spath et al., 2012), and between 2.3 × 102 and 8 × 104 CFU μg−1 transforming DNA for the Lactobacillus species (Spath et al., 2012). In this work, we investigated the effect of growth to different cell densities, either with or without glycine, and with or without 0.9 M NaCl, as well as with water or with lithium acetate/DTT pretreatment on the electrotransformation efficiencies of five strains of L. casei isolated from different environmental origins: 12A, 32G, A2-362, ATCC 334 and BL23 (Broadbent et al., 2012). Using a combined approach and optimized electroporation conditions, we obtained improved transformation efficiencies of 106 CFU μg−1 of transforming DNA or higher for three of the five strains.
MATERIALS AND METHODS Bacterial strains, plasmids and reagents Escherichia coli strain DH5α containing the pTRKH2 vector and L. casei strains 12A, 32G, A2-362, ATCC 334 and BL23 were maintained as −80◦ C glycerol stocks (15% glycerol). Vector pTRKH2 (6727 bp) carrying an S-adenosylmethionine-dependent methyltransferase gene providing erythromycin resistance is a broad host-range shuttle vector based on the theta replicon from pAMβ1 (O’Sullivan and Klaenhammer 1993). Chemicals, media and laboratory supplies were from Fisher Scientific or SigmaAldrich.
Preparation of recipient cell stocks Working cultures of L. casei (10–25 mL) were inoculated from −80◦ C stocks and grown in MRS broth at 37◦ C without shaking. Cells from these cultures were subcultured into 200 mL of prewarmed MRS broth to an optical density at 600 nm (OD600 ) of 0.1. Cultures without supplementation with glycine or with NaCl were grown at 37◦ C without shaking to OD600 ’s of 0.6, 0.8–0.9 and 1.2 (4–8 h). Cultures for glycine experiments were grown in MRS broth supplemented with 0.5 or 1.0% glycine and grown similarly to OD600 ’s of 0.6 or 0.8–0.9, while cultures for NaCl experiments were inoculated into MRS broth containing 0.9 M NaCl and grown similarly to OD600 ’s of 0.6. Cells from these cultures were harvested by centrifugation at 4◦ C using a 10 min spin at 7000 rpm in a Sorvall centrifuge with a GSA rotor, rinsed in 200 mL cold (∼4◦ C), sterile distilled water and centrifuged again. The cell pellet was gently suspended in 2–3 mL of cold sterile distilled water and 1.0 mL aliquots transferred to 1.5 mL microcentrifuge tubes. After microcentrifugation in a Hermle Z230M microcentrifuge at 15 000 rpm for 30 to 120 s, the supernatant was removed and the cells rinsed twice more with 1 mL cold sterile distilled water, and then with 1 mL of a cold sterile 30% PEG-8000 solution. The supernatant was removed and the cells in each tube suspended in 0.5–0.6 mL 30% PEG for storage at −80◦ C. These cells retained viability and transformability for periods up to two years.
Treatment of cells prior to electroporation Recipient cells were thawed at room temperature and either transformed immediately, suspended in cold sterile distilled water for 30 min or treated with a 100 mM lithium acetate and 10 mM DTT solution for 30 min. Treatment with water involved addition of 900 μL water to 600 μL cell suspension. Treatment with lithium acetate/DTT involved addition of 750 μL of lithium acetate solution (200 mM lithium acetate, 1.2 M sucrose, 20 mM Tris, pH 7.5) and 150 μL of 100 mM DTT to 600 μL cell suspension. Lithium acetate and DTT solutions were filter sterilized and stored at −20◦ C until use. The concentrations of lithium acetate, sucrose, buffer and DTT used were the same as described by Papagianni et al. (2007) and Spath et al. (2012); however, previous workers treated cells in larger volumes immediately after growth. After water or lithium acetate/DTT pretreatment, the cells were pelleted for 2–3 min in a microcentrifuge, washed once in 1 mL cold sterile 30% PEG solution and suspended in 0.5–0.6 mL cold sterile 30% PEG solution for electroporation.
Electroporation Vector pTRKH2 was prepared from E. coli cells using a cetyltrimethylammonium bromide precipitation technique (Doyle and Doyle 1987) and stored at 20 ng μL−1 in TE pH 8.0. The plasmid DNA (200 ng/transformation) was mixed with 100 μL cell suspension and transferred to a sterile prechilled electroporation cuvette (0.2 cm gap). Each electroporation contained cells from ∼10 mL of culture, a concentration factor of ∼100-fold. Using a Bio-Rad Gene Pulser (Model 1652076), the capacitance setting was 25μF with resistances of 200 or 400 and voltage settings of 1500, 2000 or 2500 V (7.5, 10.0 or 12.5 kV cm−1 ). After electroporation, 900 μL recovery medium (0.5 M sucrose in MRS broth) was added and the cells transferred to a sterile microcentrifuge tube for incubation for 4 h at 37◦ C without shaking to express erythromycin resistance. The cells were then diluted in recovery medium and plated on MRS agar containing 2.5 μg mL−1 erythromycin; typically three plates per treatment per dilution were used. Plates were incubated aerobically at 37◦ C. Erythromycin-resistant colonies were counted starting on day 2 and then for an additional 3–4 days. Cells not used immediately were stored at −80◦ C after addition of glycerol (15% final concentration); cells stored in this way yielded transformation efficiencies similar to cells plated immediately after post-electroporation incubation.
RESULTS Effects of different growth and pre-electroporation treatments The effects on transformation efficiencies of growing cells to different densities in the presence or absence of glycine and of treating cells with either a lithium acetate/DTT solution or water prior to electroporation are shown in Table 1. The five strains differed greatly in their transformation efficiencies and were affected in different ways by the altered growth conditions and treatments. Strains BL23 and 12A prepared without glycine or preelectroporation treatment transformed with efficiencies of at least 105 CFU μg−1 pTRKH2 and strain ATCC 334 transformed with efficiencies of at least 104 CFU μg−1 pTRKH2 (Table 1A). Strains 32G and A2-362 had lower transformation efficiencies of ∼102 –103 CFU μg−1 pTRKH2. Cells grown to higher cell
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Table 1. Representative transformation efficiencies in CFU μg−1 pTRKH2 using cells prepared by growth in MRS with or without glycine and with electroporation at 200 and 12.5 kV cm−1 . Condition
Strain 12A
32G
A2-362
ATCC 334
BL23
2.0 × 102 7.0 × 101 9.0 × 101 9.3 × 102 8.0 × 101 3.3 × 102
7.7 × 102 1.2 × 103 1.0 × 103 2.6 × 103 2.4 × 103 2.8 × 103
4.6 × 104 1.6 × 104 4.0 × 104 1.1 × 105 4.1 × 104 2.9 × 104
2.1 × 105 1.4 × 105 1.5 × 105 6.0 × 105 3.9 × 105 7.9 × 105
6.2 × 103 4.5 × 103 3.4 × 103 2.1 × 104 1.0 × 104 1.2 × 104
3.4 × 103 3.4 × 103 3.2 × 103 1.4 × 104 9.6 × 103 9.1 × 103
2.5 × 105 2.4 × 105 6.9 × 104 8.3 × 105 2.4 × 105 2.2 × 104
6.3 × 106 3.4 × 106 2.2 × 106 4.9 × 106 4.4 × 106 4.2 × 106
2.7 × 103 3.8 × 103 1.7 × 103 1.9 × 104 7.6 × 103 1.1 × 104
2.6 × 103 2.4 × 103 2.4 × 103 9.1 × 103 8.4 × 103 8.6 × 103
7.6 × 105 7.2 × 105 2.1 × 105 2.0 × 106 1.8 × 105 1.0 × 106
1.0 × 106 2.8 × 105 6.4 × 105 5.2 × 105 4.3 × 105 1.3 × 106
A. Without pretreatment prior to electroporation. OD600 OD600 OD600 OD600 OD600 OD600
0.6 without glycine 0.8 without glycine 1.2 without glycine 0.6 with 1% glycine 0.8 with 0.5% glycine 0.8 with 1% glycine
4.8 × 105 8.0 × 105 1.3 × 105 1.2 × 105 2.6 × 105 5.0 × 105
B. With 30 min lithium acetate/DTT pretreatment prior to electroporation. OD600 OD600 OD600 OD600 OD600 OD600
0.6 without glycine 0.8 without glycine 1.2 without glycine 0.6 with 1% glycine 0.8 with 0.5% glycine 0.8 with 1% glycine
2.6 × 106 1.4 × 106 1.2 × 106 1.6 × 106 2.6 × 106 5.4 × 106
C. With 30 min water pretreatment prior to electroporation. OD600 OD600 OD600 OD600 OD600 OD600
0.6 without glycine 0.8 without glycine 1.2 without glycine 0.6 with 1% glycine 0.8 with 0.5% glycine 0.8 with 1% glycine
2.3 × 106 2.1 × 106 2.2 × 106 2.3 × 106 2.1 × 106 4.0 × 106
densities (OD600 1.2) without glycine or pre-electroporation treatment generally had lower transformation efficiencies than cells grown to lower densities (OD600 0.6) (Table 1A). Growth of cells with 0.5 or 1% glycine yielded higher transformation efficiencies for all except strain 12A (Table 1A). Pre-electroporation lithium acetate/DTT treatment increased transformation efficiencies for most strains and growth conditions (Compare Table 1B to 1A). Strains BL23 and 12A transformed with efficiencies of 106 CFU μg−1 pTRKH2 (Table 1B), 2- to 30-fold higher than without the pretreatment. Strain ATCC 334’s transformation efficiency either remained the same or increased by up to a factor of 15-fold and ranged from 2 × 104 to 8 × 105 CFU μg−1 pTRKH2 (Table 1B); transformation efficiencies for strain 32G increased 20- to 1000-fold and for strain A2-362 increased 2- to 5-fold, both to 103 –104 CFU μg−1 pTRKH2 (Table 1B). Pre-electroporation treatment with a water incubation also increased the transformation efficiencies for most strains and growth conditions (Compare Table 1C to 1A). After water pretreatment, the transformation efficiencies for strain ATCC 334 increased 4- to 30-fold to 105 –106 CFU μg−1 pTRKH2 (Table 1C) and were higher than those obtained with lithium acetate/DTT pretreatment (Table 1B), except for cells grown to OD600 0.8 with 0.5% glycine. Transformation efficiencies for strains 12A and A2-362 increased 2- to 10-fold after water incubation and reached values similar to those seen after pretreatment with lithium acetate/DTT, while those for strain BL23 stayed the same or increased by up to 5-fold after water incubation but were lower than those seen after pretreatment with lithium acetate/DTT. Transformation efficiencies for strain 32G increased 10- to almost 1000-fold after water pretreatment but also were lower than those obtained after lithium acetate/DTT pretreatment.
The effects on transformation efficiency of different electroporation conditions The effects of different electroporation conditions involving resistances of 200 and 400 and voltages of 7.5, 10.0 and 12.5 kV cm−1 were tested. For these experiments, two sets of cells either grown to OD600 0.8 in MRS with 1% glycine or grown to OD600 0.6 in MRS with 0.9 M NaCl (Palomino et al., 2010) were used. Both sets of cells were also pretreated either with water or with lithium acetate/DTT before electroporation. The results with cells grown in 1% glycine and pretreated with water are presented in Table 2A. Strains 12A, ATCC 334 and BL23 had transformation efficiencies of 105 –106 CFU μg−1 pTRKH2 using either 200 or 400 and either 10.0 or 12.5 kV cm−1 . Their transformation efficiencies using 7.5 kV cm−1 were lower than those obtained at the same resistance settings and 10.0 or 12.5 kV cm−1 . Transformation efficiencies for strains ATCC 334 and BL23 using 12.5 kV cm−1 were lower than those obtained using 10.0 kV cm−1 , but the transformation efficiency of strain 12A was higher at 12.5 kV cm−1 than at 10.0 kV cm−1 (Table 2A). Strains 32G and A2-362 again had transformation efficiencies about 10- to 100-fold lower than seen for the other three strains but their transformation efficiencies increased as the voltage setting increased (Table 2A). Of the 15 treatments, 13 using 400 yielded higher transformation efficiencies than those with the same strain and voltage condition but with a 200 setting (Table 2A); however, more electroporations arced and therefore needed to be repeated at the 400 setting. The results obtained with the same cell preparations grown with 1% glycine but pretreated with lithium acetate/DTT are presented in Table 2B. Strains ATCC 334 and BL23 again had transformation efficiencies of 105 –106 CFU μg−1 pTRKH2 using either 200 or 400 and either 10.0 or 12.5 kV cm−1 , with 10.0 kV cm−1
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Table 2. Transformation efficiencies under different electroporation conditions using cells prepared by growth to OD600 0.8 in MRS with 1% glycine (CFU μg−1 pTRKH2, means from two transformations). Condition
Strain 12A
32G
A2-362
ATCC 334
BL23
1.1 × 103 2.2 × 103 3.6 × 103 7.4 × 102 4.1 × 103 1.3 × 104
8.8 × 104 3.0 × 105 3.4 × 105 2.4 × 105 4.7 × 105 3.6 × 105
4.4 × 105 7.5 × 105 4.0 × 105 7.9 × 105 8.8 × 105 2.8 × 105
1.6 × 103 3.2 × 103 1.1 × 104 4.1 × 103 1.5 × 104 2.0 × 104
7.4 × 104 1.9 × 105 1.9 × 105 1.3 × 105 2.5 × 105 1.6 × 105
1.1 × 106 2.9 × 106 3.9 × 106 1.9 × 106 3.8 × 106 3.2 × 106
A. With 30 min water pretreatment prior to electroporation. 200 7.5 kV cm−1 200 10.0 kV cm−1 200 12.5 kV cm−1 400 7.5 kV cm−1 400 10.0 kV cm−1 400 12.5 kV cm−1
7.3 × 104 5.1 × 105 1.0 × 106 2.3 × 105 1.2 × 106 1.8 × 106
3.9 × 102 3.8 × 103 6.9 × 103 1.5 × 103 1.2 × 104 1.2 × 104
B. With 30 min lithium acetate/DTT pretreatment prior to electroporation. 200 7.5 kV cm−1 200 10.0 kV cm−1 200 12.5 kV cm−1 400 7.5 kV cm−1 400 10.0 kV cm−1 400 12.5 kV cm−1
2.6 × 104 7.1 × 104 5.4 × 105 5.0 × 104 1.3 × 105 9.4 × 104
2.5 × 101 6.8 × 101 6.2 × 102 5.4 × 101 4.0 × 102 2.4 × 103
Table 3. Transformation efficiencies under different electroporation conditions using cells prepared by growth to OD600 0.6 in MRS with 0.9 M NaCl (CFU μg−1 pTRKH2, means from two transformations). Condition
Strain 12A
32G
A2-362
ATCC 334
BL23
8.2 × 101 2.5 × 102 1.1 × 103 4.1 × 102 1.2 × 103 2.2 × 103
4.2 × 104 1.3 × 105 3.4 × 105 1.5 × 105 4.5 × 105 1.7 × 106
4.2 × 104 1.9 × 105 3.5 × 105 1.4 × 105 3.8 × 105 6.4 × 105
1.6 × 102 1.1 × 103 1.2 × 103 1.3 × 103 5.4 × 103 4.5 × 103
1.8 × 105 3.5 × 105 8.5 × 105 3.4 × 105 1.5 × 106 2.4 × 106
3.7 × 106 6.5 × 106 1.5 × 107 9.0 × 106 1.3 × 107 1.4 × 107
A. With 30 min water pretreatment prior to electroporation. 200 7.5 kV cm−1 200 10.0 kV cm−1 200 12.5 kV cm−1 400 7.5 kV cm−1 400 10.0 kV cm−1 400 12.5 kV cm−1
1.4 × 103 3.7 × 103 1.0 × 104 1.2 × 104 3.0 × 104 2.3 × 104
2.2 × 102 3.8 × 102 8.9 × 102 2.5 × 102 1.0 × 103 4.4 × 103
B. With 30 min lithium acetate/DTT pretreatment prior to electroporation. 200 7.5 kV cm−1 200 10.0 kV cm−1 200 12.5 kV cm−1 400 7.5 kV cm−1 400 10.0 kV cm−1 400 12.5 kV cm−1
1.8 × 103 6.0 × 103 1.2 × 104 4.6 × 103 1.2 × 104 1.2 × 104
3.2 × 102 7.6 × 102 3.2 × 103 1.2 × 103 3.0 × 103 3.5 × 103
being as good as or better than 12.5 kV cm−1 . Transformation efficiencies using 7.5 kV cm−1 for these two strains were again lower than those obtained at the same resistance settings and 10.0 or 12.5 kV cm−1 . Strain A2-362 had transformation efficiencies of 103 –104 CFU μg−1 pTRKH2 with all six electroporation conditions and its transformation efficiencies increased with increasing voltage. Strain 12A had transformation efficiencies of 104 –105 CFU μg−1 pTRKH2 with all six electroporation conditions. For strain 32G, the transformation efficiencies were only 101 –103 CFU μg−1 pTRKH2, although the transformation efficiencies increased with increasing voltage. Again 13 of the 15 treatments using 400 gave higher transformation efficiencies than those with the same strain and voltage conditions but with a 200 setting (Table 2B). Strain BL23 had the highest transformation efficiencies of the five strains in the set of experiments using glycine-grown cells and its transformation efficiencies obtained after lithium acetate/DTT pretreatment were higher than those obtained after water pretreatment. Strain A2-362 also had somewhat higher
transformation efficiencies after lithium acetate/DTT pretreatment than after water pretreatment. However, strains ATCC 334, 12A and 32G had higher transformation efficiencies after water pretreatment than after lithium acetate/DTT pretreatment. A similar set of experiments was performed with the cells grown to OD600 0.6 in MRS with 0.9 M NaCl (Table 3). In these experiments transformation efficiencies increased with increasing voltage, except for three of the 400 and 12.5 kV cm−1 conditions with strains 12A and A2-362 (Table 3). Almost all electroporations using 400 yielded higher transformation efficiencies than did the electroporations at the same voltages but using 200 . Strain BL23 had the highest transformation efficiencies obtained in any experiment with multiple electroporations yielding transformation efficiencies greater than 107 CFU μg−1 pTRKH2 (Table 3B). Salt-grown BL23 cells pretreated with lithium acetate/DTT had higher transformation efficiencies (Table 3B) than glycine-grown BL23 cells pretreated with lithium acetate/DTT (Table 2B), but glycine-grown cells pretreated with water had higher transformation efficiencies (Table 2A) than
Welker et al.
salt-grown cells pretreated with water (Table 3A). Similarly, saltgrown ATCC 334 cells pretreated with lithium acetate/DTT had higher transformation efficiencies (up to 106 CFU μg−1 pTRKH2) than glycine-grown cells pretreated with lithium acetate/DTT, but glycine-grown cells pretreated with water had similar or higher transformation efficiencies than salt-grown cells pretreated with water for all but the 400 and 12.5 kV cm−1 transformations. Salt-grown 12A and A2-362 cells had lower transformation efficiencies (103 –104 and 101 –103 CFU μg−1 pTRKH2, respectively) (Table 3) than did glycine-grown cells (Table 2). Transformation efficiencies for salt-grown 32G cells pretreated with water (102 –103 CFU μg−1 pTRKH2, Table 3A) were lower than for similarly treated glycine-grown 32G cells (102 –104 CFU μg−1 pTRKH2, Table 2A). However, transformations efficiencies for salt-grown and lithium acetate/DTT pretreated 32G cells (Table 3B) were higher than for glycine-grown and lithium acetate/DTT pretreated 32G cells (Table 2B).
DISCUSSION We obtained transformation efficiencies as high as 107 CFU μg−1 pTRKH2 for strain BL23, which is 10-fold higher than the best transformation efficiency reported previously for L. casei cells transformed with a theta-replication vector (Chassy and Flickinger 1987; Luchansky et al., 1988; Natori et al., 1990; Mason et al., 2005; Palomino et al., 2010). In addition, transformation efficiencies of 106 CFU μg−1 pTRKH2 were obtained for two other strains, 12A and ATCC 334. These experiments used an approach that combined growth in MRS with 0.9 M NaCl or 1% glycine, storage at −80◦ C in 30% PEG, 30 min pre-electroporation treatments with either water or a lithium acetate/DTT solution and electroporation in 30% PEG. Transformation efficiencies of this magnitude facilitate the transfer of recombinant DNA to L. casei cells, and we have used this approach to construct gene knockouts of the cyclopropane synthase gene in strain ATCC 334 using a PheS counter-selectable marker on the vector pBS1 (Broadbent et al., 2014) and to construct additional gene knock-outs and gene replacements in strains 12A and ATCC 334 (unpublished work). No one protocol was best for all strains. For example, strains ATCC 334 and BL23 transformed with the highest efficiency when grown with 0.9 M NaCl and pretreated with lithium acetate/DTT solution but had 10- to 100-fold lower transformation efficiencies when grown with 1% glycine and pretreated with water (Tables 2 and 3). In contrast, strain 12A transformed with highest efficiency when grown with 1% glycine and pretreated with water (Tables 2 and 3). Strains 32G and A2-362 had lower transformation efficiencies than the other strains (Tables 2 and 3). Explanations for variations in transformation efficiencies are numerous and include variation in membrane lipid composition [affected by Tween 80, a component of MRS (Broadbent et al., 2014)], variation in cell wall composition and integrity [affected by growth in the presence of NaCl or glycine (Holo and Nes 1989; Palomino et al., 2013) and by treatment with lithium acetate/DTT (Papagianni et al., 2007)], and the presence or absence of capsules, incompatible endogenous plasmids or restriction endonucleases targeting the transforming DNA. Incubation with lithium acetate/DTT solution or water prior to electroporation had significant effects on transformation efficiencies. Lithium acetate/DTT treatment was previously shown to enhance transformation of L. lactis (Papagianni et al., 2007), L. plantarum and L. buchneri (Spath et al., 2012) and is presumed to work by weakening the cell wall. Incubation ‘with water’ in our
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experiments, initially performed as a control experiment, essentially dilutes the electroporation buffer, since the PEG concentration during the incubation is ∼12%. While we are unaware of others using a pre-electroporation incubation with a room temperature water or dilute PEG solution, pre-electroporation incubation of Bifidobacterium species in electroporation buffer for 2 h at 4◦ C improved transformation efficiencies by 100-fold (Serafini et al., 2012). These findings show that a combined approach employing multiple strategies for changing cell membrane fluidity, cell wall integrity and electroporation parameters can improve the transformation efficiency of L. casei. Our approach is of further usefulness in its simplicity; it avoids use of anaerobic growth conditions and it allows the easy incorporation of pre-electroporation incubations with water or lithium acetate/DTT that significantly improved transformation efficiencies. We found that recipient cells stored as long as 2 yr at −80◦ C in 30% PEG retained high viability and transformability (data not shown) which allows greater experimental flexibility and facilitates the construction of recombinant strains.
ACKNOWLEDGEMENTS We thank T.R. Klaenhammer of North Carolina State University for providing vector pTRKH2.
FUNDING This paper was supported by National Research Initiative Competitive Grant no. 2011-67009-30043 from the USDA National Institute of Food and Agriculture Program, and by the Utah Agricultural Experiment Station. This communication is approved as UAES Journal Paper Number 8723.
Conflict of Interest Peggy Steele, a member of Dr Steele’s family, is employed by DuPont Inc., a supplier of bacterial cultures to the food industry.
REFERENCES Broadbent JR, Neeno-Eckwall EC, Stahl B, et al. Analysis of the Lactobacillus casei supragenome and its influence in species evolution and lifestyle adaptation. BMC Genomics 2012;13:533. Broadbent JR, Oberg TS, Hughes JE, et al. Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on the lactic acid production by Lactobacillus casei ATCC 334 at low pH. J Ind Microbiol Biot 2014;41:545–53. Chassy BM, Flickinger JL. Transformation of Lactobacillus casei by electroporation. FEMS Microbiol Lett 1987;44:173–7. Doyle JJ, Doyle JL. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 1987;19:11–5. Holo H, Nes IF. High-frequency transformation, by electroporation, of Lactococcus lactis subsp. cremoris grown with glycine in osmotically stabilized media. Appl Environ Microb 1989;55:3119–23. Kondo JK, Johansen E. Product development strategies for foods in the era of molecular biotechnology. Anton Leeuw 2002;82:291–302. Luchansky JB, Muriana PM, Klaenhammer TR. Application of electroporation for the transfer of plasmid DNA to Lactobacillus, Lactococcus, Leuconostoc, Listera, Pediococcus, Bacillus,
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Staphylococcus, Enterococcus and Propionibacterium. Mol Microbiol 1988;2:637–46. Mason CK, Collins MA, Thompson K. Modified electroporation protocol for Lactobacilli isolated from the chicken crop facilitates transformation and the use of a genetic tool. J Microbiol Meth 2005;60:353–63. Natori Y, Kano Y, Imamoto F. Genetic transformation of Lactobacillus casei by electroporation. Biochimie 1990;72:265–9. O’Sullivan DJ, Klaenhammer TR. High- and low-copy-number Lactococcus shuttle cloning vectors with features for clone screening. Gene 1993;137:227–31. Palomino MM, Allievi MC, Grundling A, et al. Osmotic stress ¨ adaptation in Lactobacillus casei BL23 leads to structural changes in the cell wall polymer lipoteichoic acid. Microbiology 2013;159:2416–26. Palomino MM, Allievi MC, Prado-Acosta M, et al. New method for electroporation of Lactobacillus species grown in high salt. J Microbiol Meth 2010;83:164–7.
Papagianni M, Avramidis N, Filioussis G. High efficiency electrotransformation of Lactococcus lactis spp. lactis cells pretreated with lithium acetate and dithiothreitol. BMC Biotechnol 2007;7:15. Serafini F, Turroni F, Guglielmetti S, et al. An efficient and reproducible method for transformation of genetically recalcitrant bifidobacteria. FEMS Microbiol Lett 2012;333:146–52. Spath K, Heinl S, Egger E, et al. Lactobacillus plantarum and Lactobacillus buchneri as expression systems: evaluation of different origins of replication for the design of suitable shuttle vectors. Mol Biotechnol 2012;52:40–8. Spath K, Heinl S, Grabherr R. Direct cloning in Lactobacillus plantarum: electroporation with non-methylated plasmid DNA enhances transformation efficiency and makes shuttle vectors obsolete. Microb Cell Fact 2012;11:141. Wei M-Q, Rush CM, Norman JM, et al. An improved method for transformation of Lactobacillus strains using electroporation. J Microbiol Meth 1995;21:97–109.
R E S E A R C H L E T T E R – Physiology & Biochemistry
High efficiency electrotransformation of Lactobacillus casei Dennis L. Welker1,∗ , Joanne E. Hughes1 , James L. Steele2 and Jeff R. Broadbent3 1
Department of Biology, Utah State University, Logan, UT 84322-5305, USA, 2 Department of Food Science, University of Wisconsin, Madison, WI 53706, USA and 3 Department of Nutrition, Dietetics and Food Sciences, Utah State University, Logan, UT 84322-8700, USA ∗ Corresponding author: Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322-5305, USA. Tel.: 435-797-3552; Fax: 435-797-1575; E-mail: [email protected] One sentence summary: This work describes a combined approach for raising the transformation efficiency of this species that may be of use for genetically modifying other Lactobacilli. Editor: Rustam Aminov
ABSTRACT We investigated whether protocols allowing high efficiency electrotransformation of other lactic acid bacteria were applicable to five strains of Lactobacillus casei (12A, 32G, A2-362, ATCC 334 and BL23). Addition of 1% glycine or 0.9 M NaCl during cell growth, limitation of the growth of the cell cultures to OD600 0.6–0.8, pre-electroporation treatment of cells with water or with a lithium acetate (100 mM)/dithiothreitol (10 mM) solution and optimization of electroporation conditions all improved transformation efficiencies. However, the five strains varied in their responses to these treatments. Transformation efficiencies of 106 colony forming units μg−1 pTRKH2 DNA and higher were obtained with three strains which is sufficient for construction of chromosomal gene knock-outs and gene replacements. Key words: electroporation; genetic manipulation; lactic acid bacteria; transformation
INTRODUCTION Lactobacillus casei and other lactic acid bacteria (LAB) are used in numerous food, agricultural and industrial fermentations. The ability to genetically manipulate L. casei and other LAB extends the range of studies that can be undertaken to understand their physiological and biochemical properties and allows for improvement of industrial strains (Kondo and Johansen 2002). However, construction of recombinant strains is often limited by the inability to introduce DNA constructs or to generate knockout mutations and gene replacements. The introduction of recombinant DNA by electrotransformation has been widely applied for genetic manipulation of LAB.
Electroporation of L. casei was first reported over 25 years ago (Chassy and Flickinger 1987) with subsequent reports describing the application of techniques, such as growth of the cells with glycine, to improve transformation efficiency (Luchansky, Muriana and Klaenhammer 1988; Natori, Kano and Imamoto 1990; Wei et al., 1995; Mason, Collins and Thompson 2005). The best transformation efficiencies reported for L. casei with thetareplicating plasmid vectors reached 105 to 106 colony forming units (CFU) μg−1 transforming DNA (Palomino et al., 2010), compared to typical efficiencies of 102 –104 CFU μg−1 transforming DNA. Those experiments used BL23 cells grown anaerobically in Lactobacillus MRS medium supplemented with 0.9 M NaCl,
Received: 26 September 2014; Accepted: 12 November 2014 C FEMS 2014. All rights reserved. For permissions, please e-mail: [email protected]
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which has been shown to reduce the amount of cell wall lipoteichoic acid, its mean chain length and its D-alanyl substitution (Palomino et al., 2013). Another new protocol involving treatment of cells with lithium acetate and dithiothreitol (DTT) was shown to increase the electrotransformation efficiency of Lactococcus lactis spp. lactis (Papagianni, Avramidis and Filioussis 2007; Spath, Heinl and Grabherr 2012), as well as L. plantarum and L. buchneri (Spath et al., 2012). Transformation efficiencies after lithium acetate/DTT treatment ranged from 105 to 109 CFU μg−1 transforming DNA for L. lactis (Papagianni et al., 2007; Spath et al., 2012), and between 2.3 × 102 and 8 × 104 CFU μg−1 transforming DNA for the Lactobacillus species (Spath et al., 2012). In this work, we investigated the effect of growth to different cell densities, either with or without glycine, and with or without 0.9 M NaCl, as well as with water or with lithium acetate/DTT pretreatment on the electrotransformation efficiencies of five strains of L. casei isolated from different environmental origins: 12A, 32G, A2-362, ATCC 334 and BL23 (Broadbent et al., 2012). Using a combined approach and optimized electroporation conditions, we obtained improved transformation efficiencies of 106 CFU μg−1 of transforming DNA or higher for three of the five strains.
MATERIALS AND METHODS Bacterial strains, plasmids and reagents Escherichia coli strain DH5α containing the pTRKH2 vector and L. casei strains 12A, 32G, A2-362, ATCC 334 and BL23 were maintained as −80◦ C glycerol stocks (15% glycerol). Vector pTRKH2 (6727 bp) carrying an S-adenosylmethionine-dependent methyltransferase gene providing erythromycin resistance is a broad host-range shuttle vector based on the theta replicon from pAMβ1 (O’Sullivan and Klaenhammer 1993). Chemicals, media and laboratory supplies were from Fisher Scientific or SigmaAldrich.
Preparation of recipient cell stocks Working cultures of L. casei (10–25 mL) were inoculated from −80◦ C stocks and grown in MRS broth at 37◦ C without shaking. Cells from these cultures were subcultured into 200 mL of prewarmed MRS broth to an optical density at 600 nm (OD600 ) of 0.1. Cultures without supplementation with glycine or with NaCl were grown at 37◦ C without shaking to OD600 ’s of 0.6, 0.8–0.9 and 1.2 (4–8 h). Cultures for glycine experiments were grown in MRS broth supplemented with 0.5 or 1.0% glycine and grown similarly to OD600 ’s of 0.6 or 0.8–0.9, while cultures for NaCl experiments were inoculated into MRS broth containing 0.9 M NaCl and grown similarly to OD600 ’s of 0.6. Cells from these cultures were harvested by centrifugation at 4◦ C using a 10 min spin at 7000 rpm in a Sorvall centrifuge with a GSA rotor, rinsed in 200 mL cold (∼4◦ C), sterile distilled water and centrifuged again. The cell pellet was gently suspended in 2–3 mL of cold sterile distilled water and 1.0 mL aliquots transferred to 1.5 mL microcentrifuge tubes. After microcentrifugation in a Hermle Z230M microcentrifuge at 15 000 rpm for 30 to 120 s, the supernatant was removed and the cells rinsed twice more with 1 mL cold sterile distilled water, and then with 1 mL of a cold sterile 30% PEG-8000 solution. The supernatant was removed and the cells in each tube suspended in 0.5–0.6 mL 30% PEG for storage at −80◦ C. These cells retained viability and transformability for periods up to two years.
Treatment of cells prior to electroporation Recipient cells were thawed at room temperature and either transformed immediately, suspended in cold sterile distilled water for 30 min or treated with a 100 mM lithium acetate and 10 mM DTT solution for 30 min. Treatment with water involved addition of 900 μL water to 600 μL cell suspension. Treatment with lithium acetate/DTT involved addition of 750 μL of lithium acetate solution (200 mM lithium acetate, 1.2 M sucrose, 20 mM Tris, pH 7.5) and 150 μL of 100 mM DTT to 600 μL cell suspension. Lithium acetate and DTT solutions were filter sterilized and stored at −20◦ C until use. The concentrations of lithium acetate, sucrose, buffer and DTT used were the same as described by Papagianni et al. (2007) and Spath et al. (2012); however, previous workers treated cells in larger volumes immediately after growth. After water or lithium acetate/DTT pretreatment, the cells were pelleted for 2–3 min in a microcentrifuge, washed once in 1 mL cold sterile 30% PEG solution and suspended in 0.5–0.6 mL cold sterile 30% PEG solution for electroporation.
Electroporation Vector pTRKH2 was prepared from E. coli cells using a cetyltrimethylammonium bromide precipitation technique (Doyle and Doyle 1987) and stored at 20 ng μL−1 in TE pH 8.0. The plasmid DNA (200 ng/transformation) was mixed with 100 μL cell suspension and transferred to a sterile prechilled electroporation cuvette (0.2 cm gap). Each electroporation contained cells from ∼10 mL of culture, a concentration factor of ∼100-fold. Using a Bio-Rad Gene Pulser (Model 1652076), the capacitance setting was 25μF with resistances of 200 or 400 and voltage settings of 1500, 2000 or 2500 V (7.5, 10.0 or 12.5 kV cm−1 ). After electroporation, 900 μL recovery medium (0.5 M sucrose in MRS broth) was added and the cells transferred to a sterile microcentrifuge tube for incubation for 4 h at 37◦ C without shaking to express erythromycin resistance. The cells were then diluted in recovery medium and plated on MRS agar containing 2.5 μg mL−1 erythromycin; typically three plates per treatment per dilution were used. Plates were incubated aerobically at 37◦ C. Erythromycin-resistant colonies were counted starting on day 2 and then for an additional 3–4 days. Cells not used immediately were stored at −80◦ C after addition of glycerol (15% final concentration); cells stored in this way yielded transformation efficiencies similar to cells plated immediately after post-electroporation incubation.
RESULTS Effects of different growth and pre-electroporation treatments The effects on transformation efficiencies of growing cells to different densities in the presence or absence of glycine and of treating cells with either a lithium acetate/DTT solution or water prior to electroporation are shown in Table 1. The five strains differed greatly in their transformation efficiencies and were affected in different ways by the altered growth conditions and treatments. Strains BL23 and 12A prepared without glycine or preelectroporation treatment transformed with efficiencies of at least 105 CFU μg−1 pTRKH2 and strain ATCC 334 transformed with efficiencies of at least 104 CFU μg−1 pTRKH2 (Table 1A). Strains 32G and A2-362 had lower transformation efficiencies of ∼102 –103 CFU μg−1 pTRKH2. Cells grown to higher cell
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Table 1. Representative transformation efficiencies in CFU μg−1 pTRKH2 using cells prepared by growth in MRS with or without glycine and with electroporation at 200 and 12.5 kV cm−1 . Condition
Strain 12A
32G
A2-362
ATCC 334
BL23
2.0 × 102 7.0 × 101 9.0 × 101 9.3 × 102 8.0 × 101 3.3 × 102
7.7 × 102 1.2 × 103 1.0 × 103 2.6 × 103 2.4 × 103 2.8 × 103
4.6 × 104 1.6 × 104 4.0 × 104 1.1 × 105 4.1 × 104 2.9 × 104
2.1 × 105 1.4 × 105 1.5 × 105 6.0 × 105 3.9 × 105 7.9 × 105
6.2 × 103 4.5 × 103 3.4 × 103 2.1 × 104 1.0 × 104 1.2 × 104
3.4 × 103 3.4 × 103 3.2 × 103 1.4 × 104 9.6 × 103 9.1 × 103
2.5 × 105 2.4 × 105 6.9 × 104 8.3 × 105 2.4 × 105 2.2 × 104
6.3 × 106 3.4 × 106 2.2 × 106 4.9 × 106 4.4 × 106 4.2 × 106
2.7 × 103 3.8 × 103 1.7 × 103 1.9 × 104 7.6 × 103 1.1 × 104
2.6 × 103 2.4 × 103 2.4 × 103 9.1 × 103 8.4 × 103 8.6 × 103
7.6 × 105 7.2 × 105 2.1 × 105 2.0 × 106 1.8 × 105 1.0 × 106
1.0 × 106 2.8 × 105 6.4 × 105 5.2 × 105 4.3 × 105 1.3 × 106
A. Without pretreatment prior to electroporation. OD600 OD600 OD600 OD600 OD600 OD600
0.6 without glycine 0.8 without glycine 1.2 without glycine 0.6 with 1% glycine 0.8 with 0.5% glycine 0.8 with 1% glycine
4.8 × 105 8.0 × 105 1.3 × 105 1.2 × 105 2.6 × 105 5.0 × 105
B. With 30 min lithium acetate/DTT pretreatment prior to electroporation. OD600 OD600 OD600 OD600 OD600 OD600
0.6 without glycine 0.8 without glycine 1.2 without glycine 0.6 with 1% glycine 0.8 with 0.5% glycine 0.8 with 1% glycine
2.6 × 106 1.4 × 106 1.2 × 106 1.6 × 106 2.6 × 106 5.4 × 106
C. With 30 min water pretreatment prior to electroporation. OD600 OD600 OD600 OD600 OD600 OD600
0.6 without glycine 0.8 without glycine 1.2 without glycine 0.6 with 1% glycine 0.8 with 0.5% glycine 0.8 with 1% glycine
2.3 × 106 2.1 × 106 2.2 × 106 2.3 × 106 2.1 × 106 4.0 × 106
densities (OD600 1.2) without glycine or pre-electroporation treatment generally had lower transformation efficiencies than cells grown to lower densities (OD600 0.6) (Table 1A). Growth of cells with 0.5 or 1% glycine yielded higher transformation efficiencies for all except strain 12A (Table 1A). Pre-electroporation lithium acetate/DTT treatment increased transformation efficiencies for most strains and growth conditions (Compare Table 1B to 1A). Strains BL23 and 12A transformed with efficiencies of 106 CFU μg−1 pTRKH2 (Table 1B), 2- to 30-fold higher than without the pretreatment. Strain ATCC 334’s transformation efficiency either remained the same or increased by up to a factor of 15-fold and ranged from 2 × 104 to 8 × 105 CFU μg−1 pTRKH2 (Table 1B); transformation efficiencies for strain 32G increased 20- to 1000-fold and for strain A2-362 increased 2- to 5-fold, both to 103 –104 CFU μg−1 pTRKH2 (Table 1B). Pre-electroporation treatment with a water incubation also increased the transformation efficiencies for most strains and growth conditions (Compare Table 1C to 1A). After water pretreatment, the transformation efficiencies for strain ATCC 334 increased 4- to 30-fold to 105 –106 CFU μg−1 pTRKH2 (Table 1C) and were higher than those obtained with lithium acetate/DTT pretreatment (Table 1B), except for cells grown to OD600 0.8 with 0.5% glycine. Transformation efficiencies for strains 12A and A2-362 increased 2- to 10-fold after water incubation and reached values similar to those seen after pretreatment with lithium acetate/DTT, while those for strain BL23 stayed the same or increased by up to 5-fold after water incubation but were lower than those seen after pretreatment with lithium acetate/DTT. Transformation efficiencies for strain 32G increased 10- to almost 1000-fold after water pretreatment but also were lower than those obtained after lithium acetate/DTT pretreatment.
The effects on transformation efficiency of different electroporation conditions The effects of different electroporation conditions involving resistances of 200 and 400 and voltages of 7.5, 10.0 and 12.5 kV cm−1 were tested. For these experiments, two sets of cells either grown to OD600 0.8 in MRS with 1% glycine or grown to OD600 0.6 in MRS with 0.9 M NaCl (Palomino et al., 2010) were used. Both sets of cells were also pretreated either with water or with lithium acetate/DTT before electroporation. The results with cells grown in 1% glycine and pretreated with water are presented in Table 2A. Strains 12A, ATCC 334 and BL23 had transformation efficiencies of 105 –106 CFU μg−1 pTRKH2 using either 200 or 400 and either 10.0 or 12.5 kV cm−1 . Their transformation efficiencies using 7.5 kV cm−1 were lower than those obtained at the same resistance settings and 10.0 or 12.5 kV cm−1 . Transformation efficiencies for strains ATCC 334 and BL23 using 12.5 kV cm−1 were lower than those obtained using 10.0 kV cm−1 , but the transformation efficiency of strain 12A was higher at 12.5 kV cm−1 than at 10.0 kV cm−1 (Table 2A). Strains 32G and A2-362 again had transformation efficiencies about 10- to 100-fold lower than seen for the other three strains but their transformation efficiencies increased as the voltage setting increased (Table 2A). Of the 15 treatments, 13 using 400 yielded higher transformation efficiencies than those with the same strain and voltage condition but with a 200 setting (Table 2A); however, more electroporations arced and therefore needed to be repeated at the 400 setting. The results obtained with the same cell preparations grown with 1% glycine but pretreated with lithium acetate/DTT are presented in Table 2B. Strains ATCC 334 and BL23 again had transformation efficiencies of 105 –106 CFU μg−1 pTRKH2 using either 200 or 400 and either 10.0 or 12.5 kV cm−1 , with 10.0 kV cm−1
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Table 2. Transformation efficiencies under different electroporation conditions using cells prepared by growth to OD600 0.8 in MRS with 1% glycine (CFU μg−1 pTRKH2, means from two transformations). Condition
Strain 12A
32G
A2-362
ATCC 334
BL23
1.1 × 103 2.2 × 103 3.6 × 103 7.4 × 102 4.1 × 103 1.3 × 104
8.8 × 104 3.0 × 105 3.4 × 105 2.4 × 105 4.7 × 105 3.6 × 105
4.4 × 105 7.5 × 105 4.0 × 105 7.9 × 105 8.8 × 105 2.8 × 105
1.6 × 103 3.2 × 103 1.1 × 104 4.1 × 103 1.5 × 104 2.0 × 104
7.4 × 104 1.9 × 105 1.9 × 105 1.3 × 105 2.5 × 105 1.6 × 105
1.1 × 106 2.9 × 106 3.9 × 106 1.9 × 106 3.8 × 106 3.2 × 106
A. With 30 min water pretreatment prior to electroporation. 200 7.5 kV cm−1 200 10.0 kV cm−1 200 12.5 kV cm−1 400 7.5 kV cm−1 400 10.0 kV cm−1 400 12.5 kV cm−1
7.3 × 104 5.1 × 105 1.0 × 106 2.3 × 105 1.2 × 106 1.8 × 106
3.9 × 102 3.8 × 103 6.9 × 103 1.5 × 103 1.2 × 104 1.2 × 104
B. With 30 min lithium acetate/DTT pretreatment prior to electroporation. 200 7.5 kV cm−1 200 10.0 kV cm−1 200 12.5 kV cm−1 400 7.5 kV cm−1 400 10.0 kV cm−1 400 12.5 kV cm−1
2.6 × 104 7.1 × 104 5.4 × 105 5.0 × 104 1.3 × 105 9.4 × 104
2.5 × 101 6.8 × 101 6.2 × 102 5.4 × 101 4.0 × 102 2.4 × 103
Table 3. Transformation efficiencies under different electroporation conditions using cells prepared by growth to OD600 0.6 in MRS with 0.9 M NaCl (CFU μg−1 pTRKH2, means from two transformations). Condition
Strain 12A
32G
A2-362
ATCC 334
BL23
8.2 × 101 2.5 × 102 1.1 × 103 4.1 × 102 1.2 × 103 2.2 × 103
4.2 × 104 1.3 × 105 3.4 × 105 1.5 × 105 4.5 × 105 1.7 × 106
4.2 × 104 1.9 × 105 3.5 × 105 1.4 × 105 3.8 × 105 6.4 × 105
1.6 × 102 1.1 × 103 1.2 × 103 1.3 × 103 5.4 × 103 4.5 × 103
1.8 × 105 3.5 × 105 8.5 × 105 3.4 × 105 1.5 × 106 2.4 × 106
3.7 × 106 6.5 × 106 1.5 × 107 9.0 × 106 1.3 × 107 1.4 × 107
A. With 30 min water pretreatment prior to electroporation. 200 7.5 kV cm−1 200 10.0 kV cm−1 200 12.5 kV cm−1 400 7.5 kV cm−1 400 10.0 kV cm−1 400 12.5 kV cm−1
1.4 × 103 3.7 × 103 1.0 × 104 1.2 × 104 3.0 × 104 2.3 × 104
2.2 × 102 3.8 × 102 8.9 × 102 2.5 × 102 1.0 × 103 4.4 × 103
B. With 30 min lithium acetate/DTT pretreatment prior to electroporation. 200 7.5 kV cm−1 200 10.0 kV cm−1 200 12.5 kV cm−1 400 7.5 kV cm−1 400 10.0 kV cm−1 400 12.5 kV cm−1
1.8 × 103 6.0 × 103 1.2 × 104 4.6 × 103 1.2 × 104 1.2 × 104
3.2 × 102 7.6 × 102 3.2 × 103 1.2 × 103 3.0 × 103 3.5 × 103
being as good as or better than 12.5 kV cm−1 . Transformation efficiencies using 7.5 kV cm−1 for these two strains were again lower than those obtained at the same resistance settings and 10.0 or 12.5 kV cm−1 . Strain A2-362 had transformation efficiencies of 103 –104 CFU μg−1 pTRKH2 with all six electroporation conditions and its transformation efficiencies increased with increasing voltage. Strain 12A had transformation efficiencies of 104 –105 CFU μg−1 pTRKH2 with all six electroporation conditions. For strain 32G, the transformation efficiencies were only 101 –103 CFU μg−1 pTRKH2, although the transformation efficiencies increased with increasing voltage. Again 13 of the 15 treatments using 400 gave higher transformation efficiencies than those with the same strain and voltage conditions but with a 200 setting (Table 2B). Strain BL23 had the highest transformation efficiencies of the five strains in the set of experiments using glycine-grown cells and its transformation efficiencies obtained after lithium acetate/DTT pretreatment were higher than those obtained after water pretreatment. Strain A2-362 also had somewhat higher
transformation efficiencies after lithium acetate/DTT pretreatment than after water pretreatment. However, strains ATCC 334, 12A and 32G had higher transformation efficiencies after water pretreatment than after lithium acetate/DTT pretreatment. A similar set of experiments was performed with the cells grown to OD600 0.6 in MRS with 0.9 M NaCl (Table 3). In these experiments transformation efficiencies increased with increasing voltage, except for three of the 400 and 12.5 kV cm−1 conditions with strains 12A and A2-362 (Table 3). Almost all electroporations using 400 yielded higher transformation efficiencies than did the electroporations at the same voltages but using 200 . Strain BL23 had the highest transformation efficiencies obtained in any experiment with multiple electroporations yielding transformation efficiencies greater than 107 CFU μg−1 pTRKH2 (Table 3B). Salt-grown BL23 cells pretreated with lithium acetate/DTT had higher transformation efficiencies (Table 3B) than glycine-grown BL23 cells pretreated with lithium acetate/DTT (Table 2B), but glycine-grown cells pretreated with water had higher transformation efficiencies (Table 2A) than
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salt-grown cells pretreated with water (Table 3A). Similarly, saltgrown ATCC 334 cells pretreated with lithium acetate/DTT had higher transformation efficiencies (up to 106 CFU μg−1 pTRKH2) than glycine-grown cells pretreated with lithium acetate/DTT, but glycine-grown cells pretreated with water had similar or higher transformation efficiencies than salt-grown cells pretreated with water for all but the 400 and 12.5 kV cm−1 transformations. Salt-grown 12A and A2-362 cells had lower transformation efficiencies (103 –104 and 101 –103 CFU μg−1 pTRKH2, respectively) (Table 3) than did glycine-grown cells (Table 2). Transformation efficiencies for salt-grown 32G cells pretreated with water (102 –103 CFU μg−1 pTRKH2, Table 3A) were lower than for similarly treated glycine-grown 32G cells (102 –104 CFU μg−1 pTRKH2, Table 2A). However, transformations efficiencies for salt-grown and lithium acetate/DTT pretreated 32G cells (Table 3B) were higher than for glycine-grown and lithium acetate/DTT pretreated 32G cells (Table 2B).
DISCUSSION We obtained transformation efficiencies as high as 107 CFU μg−1 pTRKH2 for strain BL23, which is 10-fold higher than the best transformation efficiency reported previously for L. casei cells transformed with a theta-replication vector (Chassy and Flickinger 1987; Luchansky et al., 1988; Natori et al., 1990; Mason et al., 2005; Palomino et al., 2010). In addition, transformation efficiencies of 106 CFU μg−1 pTRKH2 were obtained for two other strains, 12A and ATCC 334. These experiments used an approach that combined growth in MRS with 0.9 M NaCl or 1% glycine, storage at −80◦ C in 30% PEG, 30 min pre-electroporation treatments with either water or a lithium acetate/DTT solution and electroporation in 30% PEG. Transformation efficiencies of this magnitude facilitate the transfer of recombinant DNA to L. casei cells, and we have used this approach to construct gene knockouts of the cyclopropane synthase gene in strain ATCC 334 using a PheS counter-selectable marker on the vector pBS1 (Broadbent et al., 2014) and to construct additional gene knock-outs and gene replacements in strains 12A and ATCC 334 (unpublished work). No one protocol was best for all strains. For example, strains ATCC 334 and BL23 transformed with the highest efficiency when grown with 0.9 M NaCl and pretreated with lithium acetate/DTT solution but had 10- to 100-fold lower transformation efficiencies when grown with 1% glycine and pretreated with water (Tables 2 and 3). In contrast, strain 12A transformed with highest efficiency when grown with 1% glycine and pretreated with water (Tables 2 and 3). Strains 32G and A2-362 had lower transformation efficiencies than the other strains (Tables 2 and 3). Explanations for variations in transformation efficiencies are numerous and include variation in membrane lipid composition [affected by Tween 80, a component of MRS (Broadbent et al., 2014)], variation in cell wall composition and integrity [affected by growth in the presence of NaCl or glycine (Holo and Nes 1989; Palomino et al., 2013) and by treatment with lithium acetate/DTT (Papagianni et al., 2007)], and the presence or absence of capsules, incompatible endogenous plasmids or restriction endonucleases targeting the transforming DNA. Incubation with lithium acetate/DTT solution or water prior to electroporation had significant effects on transformation efficiencies. Lithium acetate/DTT treatment was previously shown to enhance transformation of L. lactis (Papagianni et al., 2007), L. plantarum and L. buchneri (Spath et al., 2012) and is presumed to work by weakening the cell wall. Incubation ‘with water’ in our
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experiments, initially performed as a control experiment, essentially dilutes the electroporation buffer, since the PEG concentration during the incubation is ∼12%. While we are unaware of others using a pre-electroporation incubation with a room temperature water or dilute PEG solution, pre-electroporation incubation of Bifidobacterium species in electroporation buffer for 2 h at 4◦ C improved transformation efficiencies by 100-fold (Serafini et al., 2012). These findings show that a combined approach employing multiple strategies for changing cell membrane fluidity, cell wall integrity and electroporation parameters can improve the transformation efficiency of L. casei. Our approach is of further usefulness in its simplicity; it avoids use of anaerobic growth conditions and it allows the easy incorporation of pre-electroporation incubations with water or lithium acetate/DTT that significantly improved transformation efficiencies. We found that recipient cells stored as long as 2 yr at −80◦ C in 30% PEG retained high viability and transformability (data not shown) which allows greater experimental flexibility and facilitates the construction of recombinant strains.
ACKNOWLEDGEMENTS We thank T.R. Klaenhammer of North Carolina State University for providing vector pTRKH2.
FUNDING This paper was supported by National Research Initiative Competitive Grant no. 2011-67009-30043 from the USDA National Institute of Food and Agriculture Program, and by the Utah Agricultural Experiment Station. This communication is approved as UAES Journal Paper Number 8723.
Conflict of Interest Peggy Steele, a member of Dr Steele’s family, is employed by DuPont Inc., a supplier of bacterial cultures to the food industry.
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