Optimization of electrotransformation conditions for Leuconostoc mesenteroides subsp. mesenteroides ATCC8293

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1 Letters in Applied Microbiology ISSN ORIGINAL ARTICLE Optimization of electrotransformation conditions for Leuconostoc mesenteroides subsp. mesenteroides ATCC8293 Q. Jin,2 *, H.-J. Eom 2 *, J.Y. Jung 2, J.S. Moon 2, J.H. Kim 3 and N.S. Han 2 Department of Food Science, Yanbian University, Yanji, Jilin, China 2 Department of Food Science and Technology, BK2 Education and Research Center for Advanced Bio-Agriculture Technology, Chungbuk National University, Cheongju, Korea 3 Division of Applied Life Science (BK2), Graduate School, Gyeongsang National University, Jinju, Korea Keywords electroporation, Leuconostoc, plasmids, pleucm, transformation efficiency. Correspondence Nam Soo Han, Department of Food Science and Technology, Chungbuk National University, Cheongju , Korea. namsoo@chungbuk.ac.kr *Contributed equally to this work : received 22 May 202, revised 5 July 202 and accepted 7 August 202 doi:./j x x Abstract Aims: To establish an efficient genetic transformation protocol for Leuconostoc species, methods for competent-cell preparation and electroporation conditions were optimized. Methods and Results: Leuconostoc mesenteroides subsp. mesenteroides ATCC8293 cells were sequentially treated with penicillin G and lysozyme, and the plasmid pleucm was subsequently transformed into the cells. Our results demonstrated that transformation efficiencies were significantly increased (0-fold), and increased electric field strength also contributed to enhance transformation efficiency. Maximum transformation efficiency ( 4 or more transformants per lg DNA) was achieved when cells were grown in De Man, Rogosa, Sharpe (MRS) media containing 0Æ25 mol l ) sucrose and 0Æ8 lg ml ) penicillin G, followed by treatment with 600 U ml ) lysozyme and electroporation at a field strength of kv cm ). When this protocol was used to transform pleucm into Leuc. mesenteroides, Leuconostoc gelidum, Leuconostoc fallax and Leuconostoc argentinun, successful transformations were obtained in all cases. Furthermore, this procedure was applicable to species belonging to other genera, including Lactobacillus plantarum, Pediococcus pentosaceus and Weissella confusa. Conclusions: The results demonstrate that the transformation efficiency for Leuconostoc spp. could be increased via optimization of the entire electroporation procedures. Significance and Impact of the Study: These optimized conditions can be used for the extensive genetic study and the metabolic engineering of not only Leuconostoc spp. but also different species of lactic acid bacteria. Introduction Leuconostoc is an industrially important Gram-positive bacterium that is used as a starter culture for the manufacture of various fermented foods, such as kimchi and sauerkraut. Leuconostoc species can also be used in the production of industrial chemicals, such as mannitol and dextran (Robyt and Eklund 983; De Belder 993; Johanningsmeier et al. 2007; Eom et al. 2008). Because of this industrial importance, the genome sequence of Leuconostoc mesenteroides ATCC8293 has been first determined (Makarova et al. 2006). However, despite of its increasing attention, Leuconostoc spp. have not yet been subjected to extensive genetic engineering studies, mainly because of the absence of dedicated genetic tools. Electroporation is the most widely used technique for the easy and rapid introduction of foreign DNA into a variety of different micro-organisms. Although the molecular mechanism of electrotransformation is not completely understood, the electrical pulse is thought to result in a rearrangement of cell wall and membrane components to generate transient pores through which DNA can pass into the cell (Rixon and Warner 2003). The efficiency of electrotransformation depends on the level of electropermeabilization and pretreatment with chemicals used to alter cell wall permeability (Papagianni et al. 34 Letters in Applied Microbiology 55, ª 202 The Society for Applied Microbiology

2 Q. Jin et al. High-efficiency electrotransformation of Leuconostoc 2007). A variety of techniques have been developed to improve electrotransformation of lactic acid bacteria (LAB); treatment of cells with penicillin G (Wei et al. 995), glycine (Holo and Nes 989; Thompson and Collins 996), lysozyme (Rodríguez et al. 2007) and ethanol (Assad-García et al. 2008); application of osmotic stress (Palomino et al. 20); and optimization of several electroporation parameters (Rodríguez et al. 2007; Assad- García et al. 2008). Several electrotransformation protocols have also been reported for Leuconostoc spp., including Leuconostoc sp. (Luchansky et al. 988; David et al. 989), Leuc. mesenteroides subsp. cremoris, Leuc. mesenteroides subsp. dextranicum, Lactococcus lactis (Wyckoff et al. 99), Leuconostoc carnosum (Helmark et al. 2004) and Leuc. mesenteroides strain NRRL B-355 (Leathers et al. 2004). However, when used to transform Leuc. mesenteroides ATCC8293 in our experiments, these methods gave relatively low transformation yields ( 2 or fewer transformants per lg of plasmid DNA) and were difficult to reproduce. Therefore, we attempted to optimize an electrotransformation method for this strain by employing different treatment conditions and electroporation parameters. Here, we report the optimization of electroporation conditions for the transformation of Leuc. mesenteroides ATCC8293 using step-wise treatment of cells with lysozyme and penicillin G. For transformation, we used pleu- CM (Eom et al. 20), a shuttle vector for Leuconostoc and other LAB, and tested the applicability of the optimized protocol to other Leuconostoc strains and LAB that have potential for biotechnological applications. Materials and methods Bacterial strains, plasmid, chemicals and growth conditions Escherichia coli MC6 was grown at 37 C with shaking in Luria Bertani (LB) broth. Leuconostoc spp. (Leuc. mesenteroides ATCC8293, Leuc. mesenteroides KCTC30, Leuconostoc gelidum KCTC3527, Leuconostoc fallax KCTC3537 and Leuconostoc argentinum KCTC3773) and Weisella confusa KACC84 were grown in MRS broth (Difco, Detroit, MI, USA) at 30 C. Lactobacillus plantarum KCTC34 and Pediococcus pentosaceus KCCM902 were grown in MRS broth at 37 C. The plasmid used in this study was pleucm (5Æ8 kb), which has a broad host range with 7 plasmid copy number per Leuconostoc cell, and is used for the heterologous expression of genes in Leuconostoc species (Eom et al. 20). Plasmid DNA was isolated from E. coli MC6 using the DNA-spin Plasmid DNA Extraction Kit according to the manufacturer s protocol (Intron Biotechnology, Seongnam, Korea). Plasmid DNA was prepared from Leuconostoc sp. according to the modified method of Park et al. (997). When necessary, ampicillin (50 lg ml ) ) or chloramphenicol ( lg ml ) ) was added to the medium for the culture of E. coli or LAB, respectively. Penicillin G, lysozyme and all other chemicals were obtained from Sigma (St. Louis, MO, USA) unless otherwise stated. Electrotransformation procedure To optimize the electrotransformation conditions for Leuconostoc, the method of Wyckoff et al. (99) was modified as follows. To obtain competent cells, Leuc. mesenteroides ATCC8293 was inoculated into MRS containing different supplements and cultivated overnight at 30 C. This culture was diluted in an appropriate volume of the same medium to obtain an initial optical density at 660 nm (OD 660 )of0æ02. Cells were then grown, and samples were taken at various growth phases. After collection, the cells were placed on ice for min and harvested by centrifugation at 6000 g for min at 4 C. The cells were washed twice by resuspending the pellet in 25 ml of icecold distilled water and subsequent centrifugation at 6000 g for min at 4 C. The resulting pellet was resuspended in a small volume of ice-cold electroporation solution (EPS; 0Æ5 mol l ) sucrose, mmol l ) K 2 HPO 4 Æ KH 2 PO 4 and mmol l ) MgCl 2, ph 7Æ4) for a final OD 660 of 50. Cell aliquots were prepared and stored at )70 C. Transformation was performed using a Gene-Pulser unit combined with a Pulse Controller (Bio-Rad, Richmond, CA, USA). Cuvettes with an interelectrode distance of 0Æ cm were used. A total of 50 ll ofleuconostoc cells were mixed with 0Æ5 lg of transforming DNA in a microfuge tube, transferred to cold electroporation cuvettes and placed on ice for 5 min. A pulse was delivered under the following conditions: 25 lf, 400 X at field strengths of 2, 4, 6, 8, and 2 kv cm ). Cells were immediately resuspended in ml MRS broth containing 2% sucrose and incubated for h at 30 C. The transformed cells were spread on an MRS agar plate containing lg ml ) chloramphenicol, and plates were incubated at 30 C for 2 days until colonies were visible. For control experiments, cells that had undergone the same treatment but without the addition of plasmid were spread on selection plates. To determine the survival of Leuconostoc upon electrical shock, the cells were electroporated without plasmid DNA and plated on MRS agar without antibiotics. Confirmation of transformants Transformants were analysed by direct colony PCR. To detect the chloramphenicol acetyltransferase gene (cm) in Letters in Applied Microbiology 55, ª 202 The Society for Applied Microbiology 35

3 High-efficiency electrotransformation of Leuconostoc Q. Jin et al. the cells harbouring pleucm, forward (5 -CATATCA AATGAACTTTAAT-3 ) and reverse (5 -ATCTCATATTA TAAAAGCCA-3 ) primers were used. A single colony grown on a chloramphenicol plate was suspended in 50 ll sterilized deionized water and boiled for 5 min. One microlitre of this sample was used as template DNA. PCR amplification was carried out using a Mini Cycler (MJ Research, Waltham, MA, USA) under standard conditions (94 C for 5 min, 30 cycles of 30 s at 94 C, 30 s at 55 C, 30 s at 72 C and a final elongation at 72 C for 5 min) with 2XPCR Master Mix (Intron Biotechnology). The pleucm plasmid DNA and untransformed Leuconostoc DNA were employed as positive and negative controls, respectively. Results Effects of chemicals The effects of cell wall weakeners (glycine and threonine) and an osmotic stabilizer (sucrose) on the transformation efficiency of Leuc. mesenteroides ATCC8293 were tested while the pulse strength (8 kv cm ) ) and pulse length (400 X) were fixed. Cells were grown to an OD 660 of 0Æ6 in MRS broth supplemented with 40 mmol l ) threonine (David et al. 989), % glycine (Walker et al. 996), 0Æ25 mol l ) sucrose (Wyckoff et al. 99), 0Æ25 mol l ) sucrose + % glycine (Thompson and Collins 996) or 0Æ25 mol l ) sucrose + 40 mmol l ) threonine (Berthier et al. 996). The colonies grown on MRS agar plates containing chloramphenicol showed 667-bp PCR fragments on an agarose gel corresponding to the cm gene, revealing successful introduction of pleucm into the transformants (data not shown). As shown in Fig., 0Æ25 mol l ) sucrose-mrs medium resulted in slightly higher and more reproducible transformation efficiency among the various tested conditions, while glycine and threonine retarded cell growth. Accordingly, we used the 0Æ25 mol l ) sucrose-mrs medium in subsequent experiments for preparation of ATCC8293 competent cells. Effects of the growth phase To examine the effects of cell growth phase on transformation efficiency, samples were taken at suitable intervals from MRS broth cultures and electroporated as described above. The transformation efficiency was high (maximum 00 0 MRS + 40 mmol l Threonine MRS + % Glycine MRS mol l Sucrose + % Glycine MRS mol l Sucrose + 40 mmol l Threonine MRS mol l Sucrose Figure Effects of growth conditions on the transformation frequency of Leuconostoc mesenteroides ATCC8293 using pleucm. Data were obtained from three independent experiments; prepared competent cells were from the exponential growth phase (OD 660 =0Æ6) and electroporated at 8Æ0 kvcm ). 36 Letters in Applied Microbiology 55, ª 202 The Society for Applied Microbiology

4 Q. Jin et al. High-efficiency electrotransformation of Leuconostoc of Æ9 2 transformants per lg DNA) when cells were harvested in the middle of the exponential phase (OD 660 of 0Æ6 0Æ8). Meanwhile, cells harvested in the early exponential phase (OD 660 of 0Æ2 0Æ4) or in the late exponential phase (OD 660 of ) resulted in lower efficiencies (data not shown). Effects of lysozyme Prior to electroporation, the harvested cells were resuspended in washing buffer containing various concentrations of lysozyme and incubated at 37 C for 20 min. Cells were collected by centrifugation, washed once in ice-cold EPS and subsequently subjected to the protocol described above. Low concentration treatment with lysozyme improved the transformation efficiency; however, treatment with 2000 U ml ) decreased the reproducibility of competent cells (Fig. 2a). From this analysis, we found that treatment with 600 U ml ) lysozyme produced 3 2 transformants per lg DNA, resulting in Æ5 2 times higher yields than in non-lysozyme-treated control cells. Effects of penicillin G When cells reached an OD 660 of 0Æ2, penicillin G was added at varying concentrations and incubated for an additional 2 h until an OD 660 of 0Æ8 was reached. Cells were then harvested and electrotransformed. Transformation yields increased with the addition of 0Æ8 lg ml ) penicillin G and then decreased at higher concentrations owing to the inhibitory activity of penicillin (Fig. 2b). At the optimal concentration of penicillin G (0Æ8 lg ml ) ), 2Æ3 3 transformants were obtained per lg DNA, resulting in a 2-fold higher yield compared to untreated control cells. Effects of dual treatment of penicillin G and lysozyme When cells reached an OD 660 of 0Æ2, penicillin G was added at varying concentrations and incubated for an additional 2 h until an OD 660 of 0Æ8 was reached. Cells were harvested, treated with 600 U ml ) lysozyme and electrotransformed. Transformation yields significantly increased with sequential treatment of penicillin G and lysozyme (Fig. 3). At optimal concentrations of penicillin G (0Æ8 lg ml ) ) and lysozyme (600 U ml ) ), Æ0 4 transformants were obtained per lg DNA, a 34-fold higher yield compared to untreated control cells. Effects of electrical field strength To determine the effects of varying electrical field strength on transformation efficiencies, cells were harvested after (a) 00 0 (b) Lysozyme (Unit ml ) Penicillin (µg ml ) Figure 2 Effects of lysozyme (a) and penicillin G (b) treatments on transformation efficiencies in Leuconostoc mesenteroides ATCC8293 using pleucm. (a) Competent cells were treated with lysozyme at varying concentrations and were cultured in MRS medium containing 0Æ25 mol l ) sucrose until an OD 660 of 0Æ8 was reached. Cells were then electroporated at 8Æ0 kvcm ). (b) Cells were grown to an OD 660 of 0Æ2, and penicillin G was then added at varying concentrations. The cells were incubated for an additional 2 h until an OD 660 of 0Æ8 was reached and then electroporated at 8Æ0 kvcm ). penicillin G (0Æ8 lg ml ) ) and lysozyme (600 U ml ) ) treatments and were electroporated at various voltages (constant 400 X and 25 lf). As shown in Fig. 4, transformation yields increased when the electric field strength increased, and the maximum yield (Æ5 4 transformants per lg DNA) was achieved at kv cm ). Optimized protocol for electroporation of Leuconostoc mesenteroides ATCC8293 On the basis of the results obtained in the above experiments, we developed an improved electrotransformation protocol for Leuc. mesenteroides ATCC8293 as described Letters in Applied Microbiology 55, ª 202 The Society for Applied Microbiology 37

5 High-efficiency electrotransformation of Leuconostoc Q. Jin et al Penicillin (µg ml ) Figure 3 Effects of sequential treatment with penicillin G and lysozyme on the transformation efficiency of Leuconostoc mesenteroides ATCC8293 using pleucm. Cells were grown to an OD 660 of 0Æ2, and penicillin G was added at varying concentrations. The cells were then incubated for an additional 2 h until an OD 660 of 0Æ8 was reached, treated with 600 U ml ) lysozyme and electroporated at 8Æ0 kvcm ) Electric field strength (kv cm ) Figure 4 Effects of electrical field strength on the transformation efficiency of Leuconostoc mesenteroides ATCC8293 using pleucm. Cells were cultured in MRS medium containing 0Æ25 mol l ) sucrose until an OD 660 of 0Æ8 was reached, treated with 0.8 lg ml ) penicillin G and 600 U ml ) lysozyme and electroporated at various electrical field strengths. below. A fresh culture of strain ATCC8293 was inoculated into 0 ml MRS broth and grown for 2 3 h to reach an OD 660 of 0Æ2. Penicillin G was added at a final concentration of 0Æ8 lg ml ), and cells were cultured to reach an OD 660 of 0Æ8. Cells were harvested, washed with distilled water and resuspended in 5 ml EPS. Lysozyme was added to a final activity of 600 U ml ) and incubated at 37 C for 20 min. Cells were then washed twice and resuspended in ice-cold EPS for a final OD 660 of 50. Next, 50 ll of competent cells were mixed with 0Æ5 lg of transforming DNA in a microfuge tube, transferred to cold electroporation cuvettes and placed on ice for 5 min. A pulse was applied under the following condition: 25 lf, 400 X and kv cm ). The cells were immediately resuspended in ml MRS broth containing 2% sucrose and incubated for h at 30 C. Transformed cells were spread on an MRS agar plate containing antibiotics ( lg ml ) chloramphenicol). Plates were incubated at 30 C for 2 days until colonies were visible. As shown in Fig. 5, the newly optimized procedure increased the transformation efficiency of Leuc. mesenteroides ATCC8293 from 2 to 4 transformants per lg DNA by a step-wise optimization of each treatment. Transformation of 3 LAB species Using the conditions optimized for Leuc. mesenteroides ATCC8293, transformation efficiencies were examined for other species of Leuconostoc and 3 LAB species, such as Leuc. mesenteroides, Leuc. gelidum, Leuc. fallax, Leuc. argentinum, Lact. plantarum, Ped. pentosaceus and W. confusa. As shown in Table, a variety of transformation efficiencies were observed among the tested strains. Interestingly, Leuc. argentinum showed the highest transformation efficiency (5Æ6 4 transformants per lg DNA) among the tested strains. Discussion In this study, through the optimization of electrotransformation conditions, we achieved an enhancement in electrotransformation efficiency in Leuc. mesenteroides ATCC8293. The transformation efficiencies of other strains of Leuconostoc and 3 LAB species under optimum conditions were also examined. Of the many factors that affect electroporation efficiencies, the structure and density of the cell wall is critical (Powell et al. 988). In particular, gram-positive bacteria with thick cell walls exhibit lower transformation efficiencies than gram-negative bacteria (Trevors et al. 992). Permeation of the rigid gram-positive cell wall could be achieved by chemically or enzymatically weakening the cell wall prior to electrotransformation. A number of investigations have attempted to weaken the cell wall through the addition of glycine or threonine and by treating cells with lysozyme or penicillin (Wei et al. 995; Helmark et al. 2004; Rodríguez et al. 2007). The effects of cell wall weakening agents, however, are not universal; these agents were found to be either highly strain specific (Eynard and Teissie 2000) or completely ineffective in some micro-organisms (Berthier et al. 996). According to Holo and Nes (989), the transformation efficiency of L. lactis increased exponentially with increasing glycine concentrations in the range of 0Æ5 2% and in the presence of sucrose. For Leuc. carnosum, the 38 Letters in Applied Microbiology 55, ª 202 The Society for Applied Microbiology

6 Q. Jin et al. High-efficiency electrotransformation of Leuconostoc Figure 5 Effects of treatments employed in this study on the transformation efficiency of Leuconostoc mesenteroides ATCC8293 using pleucm. Cells were cultured in MRS medium containing 0Æ25 mol l ) sucrose until an OD 660 of 0Æ8 was reached, treated with 0Æ8 lg ml ) penicillin G and 600 U ml ) lysozyme and electroporated at Æ0 kvcm ). MRS OD 0 8 mol l Sucrose Lysozyme 600 Unit ml Penicillin Lysozyme 0 8 µg ml 600 Unit ml + Penicillin 0 8 µg ml Voltage KV Table Transformation frequencies of Leuconostoc strains and other lactic acid bacteria using pleucm Organisms Transformation frequency (CFU lg ) DNA)* Leuconostoc mesenteroides ATCC8293 (Æ5 ±0Æ22) 4 Leuconostoc gelidum KCTC3527 (Æ2 ±0Æ2) 3 Leuc. mesenteroides KCTC30 (Æ0 ±0Æ0) 3 Leuconostoc fallax KCTC3537 (Æ ±0Æ0) 3 Leuconostoc argentinum KCTC3773 (5Æ6 ±2Æ24) 4 Leuconostoc citreum KM20 (Æ4 ±0Æ0) 3 Lactobacillus plantarum KCTC34 (Æ4 ±0Æ00) 2 Pediococcus pentosaceus KCCM902 (3Æ7 ±0Æ00) 0 Weissella confusa KACC84 (Æ9 ±0Æ00) *Expressed as chloramphenicol-resistant colonies recovered per microgram of pleucm isolated from Escherichia coli (average of three independent experiments). Electroporation was conducted by following the optimized protocol obtained in this study. highest transformation efficiency was achieved when 0Æ5 Æ0% glycine was added to the growth medium (Helmark et al. 2004). Our results demonstrated that transformation yields were slightly improved in sucrose-containing medium, and this may have resulted from the osmotic stabilizing effects of sucrose and or the protective effects of dextran synthesized by Leuc. mesenteroides in the presence of sucrose (Robyt and Eklund 983). According to previous reports, treatment of LAB cells with different concentrations of lysozyme prior to electroporation improved transformation efficiencies (Wei et al. 995; Rodríguez et al. 2007). Consistent with this study, the step-wise treatment of ATCC8293 cells with penicillin G and lysozyme before electroporation resulted in an enhancement of transformation yields by up to 2 3 log units compared to the transformation yield of untreated cells. No previous reports have described the pretreatment of Leuconostoc strains with penicillin G and lysozyme. Electrical parameters are also important for altering cell wall structure. Electrical pulses introduce pores in the bacterial cell membrane and allow for the penetration of foreign DNA into cells. Electroporation at various pulses (0 Æ0 kvcm ) ) resulted in a significant increase in the number of transformants, indicating that higher voltages may give rise to an even larger number of transformants (Fig. 4). In a previous study, Leuc. mesenteroides subsp. cremoris was transformed optimally at 8 kv cm ), but poorly at kv cm ) (Wyckoff et al. 99). With regard to other species, L. paramesenteroides (now considered W. paramesenteroides) was optimally transformed at 6Æ25 kv cm ) (David et al. 989) and Leuc. mesenteroides NRRL B-355 was transformed at kv cm ) (Leathers et al. 2004). Host types also play an important role in transformation efficiency. When the optimized protocol was applied to other Leuconostoc spp., transformation yields varied among species, and Leuconostoc argentinun in particular showed the best result (5Æ6 4 transformants per lg DNA; Table ). It has been proposed that these variations may be caused by incompatibilities between foreign and endogenous plasmids, differences in restriction modification systems of the host and plasmid sizes (Posno et al. 99; Trevors et al. 992; Serror et al. 2002). Letters in Applied Microbiology 55, ª 202 The Society for Applied Microbiology 39

7 High-efficiency electrotransformation of Leuconostoc Q. Jin et al. In conclusion, this study describes an electrotransformation protocol for the transformation of pleucm into Leuc. mesenteroides ATCC8293 that is reproducible and convenient, and also the protocol may be applicable to other LAB strains. These optimum conditions can be used for the expression of heterologous proteins using pleu- CM in Leuc. mesenteroides ATCC8293. This method will allow for the introduction of a variety of metabolic functions and genetic manipulations for future strain development and biological applications of Leuc. mesenteroides ATCC8293. Acknowledgements This work was supported by Korean Research Foundation grant ( ) funded by the Korea government (MEST) and National Natural Science Foundation of China (project no ). References Assad-García, J.S., Bonnin-Jusserand, M., Garmyn, D., Guzzo, J., Alexandre, H. and Grandvalet, C. 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