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Applied and Environmental Microbiology, June 2004, p. 3695-3699, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3695-3699.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Transformation of Leuconostoc carnosum 4010 and Evidence for Natural Competence of the Organism

Søren Helmark,¹ Mette E. Hansen,¹ Birthe Jelle,² Kim I. Sørensen,² and Peter R. Jensen^1*

BioCentrum, Technical University of Denmark, DK-2800 Lyngby,¹ Chr. Hansen A/S, DK-2970 Hørsholm, Denmark²

Received 26 February 2004/ Accepted 27 February 2004

Top Abstract Introduction Materials and Methods Results Discussion References

 
Plasmid transformation in Leuconostoc carnosum 4010 was analyzed. A successful transformation protocol for L. carnosum was established by modifying an existing protocol for Lactococcus lactis. Several parameters, including the number of generations that the cells had grown at the time of harvest, glycine concentration, the time of incubation for phenotypic expression, and the electrical field strength, were investigated and proved to have influence on the transformation frequency. Electrocompetence was found to be transient and to peak in the early exponential growth phase. Optimized conditions resulted in transformation frequencies of up to 6.7 x 10⁵ transformants per microgram of plasmid DNA. A total of five plasmids in L. carnosum were successfully introduced and maintained. Interestingly, we discovered that DNA uptake was of a frequency of 3 x 10^–6 to 19 x 10^–6 transformants per CFU in the absence of an applied electrical field. We concluded that L. carnosum is naturally competent.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lactic acid bacteria (LAB) have the potential to preserve foods and have been used for this purpose for thousands of years. One important attribute of LAB is their ability to produce several metabolic products like organic acids, fatty acids, hydrogen peroxide, and diacetyl that have antimicrobial effects (4, 13, 15). In recent years interest has been paid to the ability of LAB to produce antimicrobial compounds called bacteriocins. These compounds have potential as natural substitutes for chemical food preservatives in the production of foods with an enhanced shelf life (3, 18).

Leuconostoc species are widespread in the natural environment and are of technological interest in many different food industries due to their advantageous fermentation characteristics (6). Leuconostoc carnosum is a facultative anaerobic, psychrotrophic LAB, originally associated with spoiled, packaged-meat products (1, 8, 16). However, investigations have revealed that this spoilage organism is a powerful inhibitor of bacterial growth due to its production of bacteriocins, with particularly strong antagonistic activity against the food-borne pathogen Listeria monocytogenes (2).

Advances in genetic technologies have made it possible to develop LAB starter cultures with enhanced fermentation characteristics, and today the genetic manipulation of LAB through the introduction of plasmid DNA has many potential applications in the development of improved food products. Electroporation has been used to transfer DNA into a variety of different LAB as an easy and rapid method compared to most other methods for the transfer of plasmid DNA to bacteria. Protocols for the electroporation of Leuconostoc have been described (5, 12), and in 1991 these protocols were modified by Wycloff et al. (19) for the electroporation of Leuconostoc mesenteroides subsp. cremoris, L. mesenteroides subsp. dextranicum, and Leuconostoc lactis. However, until now, no protocol has been described for L. carnosum.

The aim of the present study was to develop a DNA transformation protocol for L. carnosum. We show that up to 6.7 x 10⁵ transformants per µg of plasmid DNA could be achieved by the careful adjustment of parameters. Furthermore, we provide evidence that L. carnosum is naturally competent and takes up plasmid DNA also in the absence of an electrical field.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strain and plasmids.
L. carnosum 4010 (2) was kindly provided by Chr. Hansen A/S, Hoersholm, Denmark. This strain has been isolated and patented by The Danish Meat Research Institute, Roskilde, Denmark, and is approved by the Danish authorities for bioprotective use (2). The five plasmids applied in this study are listed in Table 1.

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TABLE 1. Plasmids used the the present study

Media.
L. carnosum was grown at 25°C in brain heart infusion (BHI) broth (37 g/liter of broth; Oxoid). For agar plates, BHI agar (47 g/liter of agar; Oxoid) was applied, and the plates were incubated at 25°C for 3 days. For the selection of plasmid-containing transformants, BHI agar was supplemented with either 3 µg of erythromycin per ml or 5 µg of chloramphenicol or tetracycline per ml, depending on the applied plasmid.

Transformation protocol.
L. carnosum was transformed by the method of Holo and Nes (10) developed for Lactococcus lactis subsp. cremoris with the following modifications. To obtain competent cells, L. carnosum was grown overnight in SGBHI, a solution containing BHI broth supplemented with 0.25 M sucrose, 27.8 mM glucose, and 1% (wt/vol) glycine. This culture was diluted in an appropriate volume of the same solution to get an initial optical density at 620 nm (OD₆₂₀) of 0.03 to 0.04. Growth was followed, and at various times samples were taken, and the cells were put on ice for 2 min and centrifuged (for 5 min at 5,000 x g and 4°C). The cells were washed in ice-cold washing solution containing 0.25 M sucrose and 10% (wt/vol) glycerol, resuspended in a small volume of the washing solution corresponding to an OD₆₂₀ of 30, and either frozen for later use or electroporated directly.

For electroporation, 50 µl of cell suspension was mixed with 0.05 µg of plasmid DNA and transferred to a sterile prechilled Gene Pulser cuvette (Bio-Rad Laboratories, Richmond, Calif.) with an interelectrode distance of 0.2 cm. The cuvette was left to stand on ice for 5 min prior to electroporation. The pulse was delivered by a Gene Pulser (Bio-Rad Laboratories) under the following conditions: 25 µF, 200 , and various voltages. The cells were immediately resuspended to a final volume of 1 ml in a room temperature resuspension solution containing BHI broth supplemented with 0.25 M sucrose, 27.8 mM glucose, 20 mM MgCl₂, and 2 mM CaCl₂ and incubated at 25°C for 2 h to allow for the phenotypic expression of the antibiotic resistance marker. The cells were centrifuged (for 2 min at 4,000 x g) and resuspended in 1 ml of 0.9% (wt/vol) NaCl, and appropriate dilutions were plated on selective BHI agar plates. Transformants were enumerated after 3 days of incubation at 25°C.

For control experiments, cells that had been given the same treatment but had received no plasmid were plated on selective plates. To determine the survival of L. carnosum upon electrical shock, the cells were electroporated without plasmid DNA and plated on BHI agar without antibiotics.

DNA isolation and analysis.
Antibiotic-resistant transformants were grown overnight in BHI broth containing the appropriate antibiotic. Plasmid DNA was isolated by using a QIAGEN very-low-copy-number plasmid purification kit, with the modifications that buffer P1 (50 mM Tris-HCl [pH 8.0], 10 mM EDTA, 100 µg of RNase A/ml) was supplemented with 20 mg of lysozyme per ml and the cells were incubated in this solution for 15 min at 37°C before the addition of buffer P2 (200 mM NaOH, 1% [wt/vol] sodium dodecyl sulfate). Agarose gel electrophoresis was applied for the analysis of the plasmid profiles of the L. carnosum transformants.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Electroporation of plasmid DNA in L. carnosum.
The major objective of this study was to develop a transformation protocol for L. carnosum. We first used a protocol described by Wycloff et al. in 1991 for the electroporation of L. mesenteroides subsp. cremoris, L. mesenteroides subsp. dextranicum, and L. lactis (19). According to this protocol, cells are grown to mid-log phase in a medium supplemented with threonine. However, this protocol yielded no transformants of L. carnosum (data not shown). We then tested a protocol developed by Holo and Nes (10) for the electroporation of Lactococcus lactis subsp. cremoris. According to this protocol, cells are grown in a medium supplemented with 0.25 M sucrose, 27.8 mM glucose, and 1% (wt/vol) glycine.

In an initial experiment we used the protocol optimized for Lactococcus lactis subsp. cremoris in order to transform L. carnosum with plasmid pCI372, a shuttle vector for Escherichia coli and Lactococcus lactis (9). An electrical pulse of 5.0 kV/cm was applied and a transformation frequency of 4.4 x 10⁵ transformants per µg of DNA was achieved. Cells that had been electroporated in the absence of DNA did not produce colonies resistant to chloramphenicol. The transformation was verified by agarose gel electrophoresis of the plasmid DNA isolated from one of the transformants, and restriction enzyme analysis confirmed the presence of pCI372.

However, repeating the experiment, we observed that the yield of transformants varied significantly, and for that reason we decided to characterize which parameters were critical for transformation efficiency and for optimizing the protocol. To determine the optimal conditions for electroporation of L. carnosum, the strain was electroporated under different conditions by using the same DNA vector, pCI372. The parameters evaluated were (i) the growth phase prior to harvest, (ii) the concentration of glycine in the growth medium, (iii) the incubation time for phenotypic expression of the antibiotic resistance marker, and (iv) the electrical field strength. Whether electrocompetent cells could be frozen and stored for later use was also investigated.

Effect of the growth phase.
Three experiments, designated A, B, and C, were performed in order to evaluate the effect of the number of generation times on the transformation frequency, and this proved to be a very critical parameter (Fig. 1.). Cells of an overnight culture in the stationary phase were inoculated in fresh SGBHI with 1% glycine to initial OD₆₂₀ values of 0.039, 0.033, and 0.063 in experiments A, B, and C, respectively, and in the three experiments, the yield of transformants varied significantly with the time of growth (Fig. 1.). If cells from the stationary overnight culture were electroporated directly, no transformants were obtained; and when the cells were allowed to grow in the fresh medium, the yield of transformants increased dramatically until an optimum after one to two generation times, which was in the early exponential growth phase.

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FIG. 1. Effect of the number of generations of cell growth at the time of harvest. The cells were grown with 1% (wt/vol) glycine in SGBHI and electroporated at 5.0 kV/cm. Cells were inoculated to an initial OD620 of 0.039 (A), 0.033 (B), and 0.063 (C).

In experiments A, B, and C, this optimum was found within a rather narrow window, i.e., from 1.1 to 1.9 generations, while the actual cell density appeared to be a less critical parameter for the development of electrocompetence in L. carnosum.

The highest transformation frequency observed was 6.7 x 10⁵ transformants per µg of DNA after 1.9 generations (Fig. 1, experiment C). After reaching the maximum yield of transformants after one to two generations, the transformation frequency decreased, and in two out of the three experiments, zero transformants were obtained after either 2.9 or 3.8 generations, although the cells were still in the exponential growth phase at this point. In experiment B, the yield of transformants did not drop to zero, probably because the experiment was stopped before reaching this point.

These findings indicate the importance of harvesting cells for electrotransformation in the early exponential growth phase in order to obtain the optimal transformation frequency of L. carnosum. In fact, the time of growth before harvest is an absolutely critical parameter, since cells in the late exponential phase were not transformable at all.

Effect of glycine concentration.
The protocol employed in this study was originally developed for Lactococcus lactis, and one of the critical parameters for the transformation efficiency of Lactococcus lactis was found to be the glycine concentration (10). The effect on the transformation frequency of L. carnosum of changing the glycine concentration is shown in Fig. 2. Transformants were obtained with and without the addition of glycine to SGBHI, but when 0.5 to 1.0% glycine was added to the medium, a maximum of 3.0 x 10⁵ to 3.2 x 10⁵ transformants per µg of DNA was achieved.

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FIG. 2. Effect of glycine concentration on transformation frequency of L. carnosum. Cells were grown in SGBHI supplemented with up to 2% (wt/vol) glycine and electroporated at 5.0 kV/cm.

Effect of phenotypic expression.
Experiments were performed to examine the effect on the transformation frequency of the duration of the incubation time for the phenotypic expression of the antibiotic resistance marker. The transformation frequency increased with the duration of the incubation time (Fig. 3), and it was decided to incubate the cells in subsequent experiments for 2 h for the phenotypic expression of the antibiotic marker.

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FIG. 3. Effect of phenotypic expression time on transformation frequency. Cells were grown with 1% (wt/vol) glycine in SGBHI and electroporated at 5.0 kV/cm.

Effect of electrical field strength.
Prior to investigating the effect of the electrical field strength on the transformation frequency, the resistance of L. carnosum to electrical shock in the absence of plasmids was determined. Cells were submitted to various electrical field strengths ranging from 2.5 to 12.5 kV/cm, and the survival percentage was calculated relative to cells that had received no electrical pulse. This test showed that virtually no cells of L. carnosum were killed when voltages below 7.5 kV/cm were applied. Sixty-five percent of the cells survived when a voltage of 10.0 kV/cm was applied, and 40% of the cells survived when 12.5 kV/cm was applied (data not shown).

The effect of the electrical field strength on the transformation frequency is shown in Fig. 4. Transformants could be detected at all the voltages applied, with a maximum of approximately 4.5 x10⁵ transformants per µg of DNA when 2.5 to 5.0 kV/cm was applied. Surprisingly, a frequency of 4.5 x 10^–6 transformants per CFU, which corresponds to 1.4 x 10⁵ transformants per µg of DNA, was obtained at 0.0 kV/cm, showing that L. carnosum has the ability to take up plasmid DNA without being exposed to an electrical pulse.

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FIG. 4. Effect of the electrical field strength on transformation frequency of L. carnosum. Cells were grown with 1% (wt/vol) glycine in SGBHI.

Transformation of frozen stored cells.
Experiments were performed with electrocompetent cells of L. carnosum that had been stored in the electroporation buffer at –80°C for 15 days (Table 2). The efficiency of the electroporation was slightly decreased during storage, with results of from 3.7 x 10⁵ to 2.5 x 10⁵ transformants per µg of DNA for cells grown in SGBHI with 1% glycine and from 2.6 x 10⁵ to 0.6 x 10⁵ transformants per µg of DNA for cells grown in the absence of glycine. However, the cells were able to take up DNA without being exposed to an electrical field, yielding 19 x 10^–6 transformants per CFU (corresponding to 4.6 x 10⁵ transformants per µg of DNA) for cells grown in SGBHI with glycine and 3.4 x 10^–6 (corresponding to 0.8 x 10⁵ transformants per µg of DNA) for cells grown in the absence of glycine.

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TABLE 2. Effect of storage on transformation frequencies of L. carnosuma

Transformation efficiency of other plasmids.
We also tested transformation with other plasmids in L. carnosum by using the optimized conditions for the electroporation (i.e., transformation at 5.0 kV/cm, the presence of 1% glycine in the growth medium, harvest after one or two generations, and selection for the proper antibiotic marker).

A total of five different plasmids representing three different replicons were introduced in L. carnosum (Table 1). Four of them, pCI372, pCI3340, pG⁺host8, and pG⁺host9:ISS1, yielded similar transformation frequencies of up to 3.0 x 10⁵ transformants per µg of DNA in frozen cells (data not shown). Only plasmid pAK80 transformed with a lower frequency of 3.6 x 10³ transformants per µg of DNA, possibly due to its rather large molecular size of 11.0 kb. The presence of plasmids in the transformants was confirmed by agarose gel electrophoresis of the plasmid preparations.

Further investigations of the natural competence of L. carnosum.
L. carnosum was also transformed directly on an agar plate. A total of 0.1 ml of an overnight culture in BHI or SGBHI was plated on an agar plate of BHI or SGBHI, respectively, and 10 µl of a solution of plasmid pCI327 (0.05 µg/ml) was immediately added to marked spots on the agar surface. After various incubation times (0, 1, 2, 3, 4, 8, and 24 h), cells were streaked from the marked spots and onto selective agar plates (i.e., BHI agar with 5 µg of chloramphenicol/ml), and these plates were incubated for 3 days. A total of four transformants were found on the selective plates in this experiment, two transformants originating from the BHI agar and two transformants originating from the SGBHI agar, and all four transformants were found after 8 h of incubation. The presence of the plasmids was again confirmed by agarose gel electrophoresis of the plasmid preparations.

Top Abstract Introduction Materials and Methods Results Discussion References

 
The successful transformation of L. carnosum was established by modifying an electroporation protocol for Lactococcus lactis developed by Holo and Nes (10). In the initial experiments various yields of transformants were obtained, and for that reason the critical parameters, i.e., the time of harvest, glycine concentration, incubation time, and electrical field strength were investigated in order to optimize the protocol for L. carnosum.

It was found that the number of generations that the cells had grown at the time of harvest was a very critical parameter for transformation efficiency. In fact, electrocompetence in L. carnosum seems to be an inducible phenomenon, similar to natural competence in other gram-positive bacteria such as Bacillus subtilis and Streptococcus pneumoniae (7, 17). Thus, it appears to be important to harvest L. carnosum cells after growth for only one to two generations after dilution of an overnight culture in order to obtain a high transformation frequency. Allowing the cells to grow for more generations will, of course, increase the number of cells but will reduce the overall transformation frequency. No transformants were obtained from the transformation of cells harvested in the stationary growth phase. L. carnosum is, thus, only electrocompetent in the early exponential growth phase.

Glycine is known to weaken cell walls, and we tested whether the amount of glycine in the growth medium would affect the transformation frequency of L. carnosum. According to Holo and Nes (10), the transformation frequency of Lactococcus lactis increased exponentially with increasing glycine concentrations in the range of 0.5 to 2% and in the presence of sucrose. For L. carnosum, the highest transformation frequency was achieved when 0.5 to 1.0% glycine was added to the growth medium, and the addition of 2.0% glycine resulted in decreased transformation frequency. Cells that had grown in SGBHI without the addition of glycine were also transformable by electroporation.

The incubation time for phenotypic expression was also optimized, and it was decided to allow 2 h for phenotypic expression at 25°C. Allowing a longer incubation time would give rise to a higher number of colonies; however, this could be due to the formation of sister transformants arising from an initially single transformant.

By using optimized conditions (i.e., the cells were grown in the presence of 1% glycine, harvested after approximately 1.9 generation times, and incubated for 2 h at 25°C prior to plating on selective plates), a maximum of 6.7 x 10⁵ transformants per µg of DNA were obtained with the plasmid pCI372 when the cells where electroporated at 5.0 kV/cm. Under these conditions, five different plasmids (Table 1), ranging in size from 4.4 to 11.0 kb, were successfully introduced in L. carnosum.

Interestingly, L. carnosum also took up plasmid DNA without being exposed to an electrical pulse, and the transformation frequency was only slightly lower than that achieved when an electrical pulse was applied, suggesting that L. carnosum has a natural competence state.

Cells stored for up to 15 days were also transformable by electroporation, yielding similar numbers of transformants before and after storage. Transformants were also achieved from stored cells without the application of an electrical pulse. In fact, higher numbers of transformants were achieved from natural transformation than from electrotransformation: 4.6 x 10⁵ transformants per µg of DNA for the stored cells compared to 2.5 x 10⁵ transformants per µg of DNA for the cells grown in the presence of glycine, indicating that natural transformation might be the most effective means of transformation of stored cells. The fact that cells that had grown in the absence of glycine and had received no electrical pulse yielded transformants (3.4 x 10^–6 transformants per CFU) provided strong evidence that L. carnosum is naturally competent.

Natural competence did not only occur in liquid media but was also observed on agar plates, where a total of four transformants were identified. Natural transformation on agar plates could be an easy-to-use means for the introduction of genetic material into L. carnosum, but the rather low number of transformants achieved in this way suggests that this might only be possible with plasmids that transform with high efficiency.

The findings that five plasmids of Lactococcus origin could be established in L. carnosum and that L. carnosum is naturally competent indicate that genetic material can be transferred to L. carnosum by horizontal gene transfer in nature. Moreover, the results also showed that three selectable markers could be used for L. carnosum: chloramphenicol, erythromycin, and tetracycline.

A database search for competence genes returned a species of Leuconostoc, L. mesenteroides subsp. mesenteries ATCC 8293. This strain has three competence genes, a negative regulator for competence and sporulation, a DNA-RNA helicase required for competence, and the competence gene comGC, suggesting that natural competence might be a more widely spread phenomenon within the Leuconostoc genus.

The state of natural competence of L. carnosum could be used as a molecular biological tool for the transfer of DNA to this organism. However, natural competence in L. carnosum needs to be further explored, and in the meantime, the described protocol for electrotransformation of L. carnosum can be used as a reliable means for the introduction of genetic material. This method will allow the introduction of a variety of different metabolic functions, which in turn will allow future molecular biological studies of L. carnosum.

 
We thank Brian J. Koebmann, Christian Solem, and Regina Schürmann for technical guidance and help in the laboratory and Alexandra Gruss, INRA, Jouy-en-Josas, France, for critical comments on the manuscript.

This work was supported by Chr. Hansen A/S and the Center for Advanced Food Studies (LMC).

 
* Corresponding author. Mailing address: BioCentrum, Technical University of Denmark, Building 301, DK-2800 Lyngby, Denmark. Phone: 45 45252510. Fax: 45 45932809. E-mail: PRJ{at}BioCentrum.DTU.DK.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Applied and Environmental Microbiology, June 2004, p. 3695-3699, Vol. 70, No. 6
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.6.3695-3699.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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