Transformation of Lactobacillus strains used in meat and vegetable fermentations

Food Research International 25 (1992) 253-261

Transformation of Lactobacihs strains used in meat and vegetable fermentations* Thea Aukrust & Hans Blom MATFORSK,

Norwegian Food Research Institute, Osloveien 1, N-14300, Aas, Norway

For transformation of Lactobacillus plantarum and Lactobacillus sake strains of meat and vegetable origin, electroporation using the electroporation solutions 952 mM sucrose with 3.5 mM MgCl, or 30% polyethylene glycol (PEG 1500) was found to be efficient. The highest transformation efficiencies for all the meat strains and most of the vegetable strains were obtained using PEG 1500. Although the transformation efficiencies varied, the strains tested showed essentially the same response pattern to different electrical pulses, as illustrated by response surface plots. For screening purposes a setting of 1.5 kV and 400 ohm pulse control resistance is recommended. For optimization of transformation efficiency for specific strains, selected combinations of voltage and pulse control resistance along the optimum shown in the plots can be used. Transformation efficiencies can be further improved by using medium shift from MRS without dextrose to MRS, and by including a magnesium solution in the washing procedure. The combined effect of these pre-treatments has raised the transformation frequency of previously low transforming strains with a factor of 103, giving transformation efficiencies sufficient for most practical applications. Keywords: Lactobacillus, electroporation,

strain variation, growth conditions.

INTRODUCTION

ing fermentation, genetically transformed strains represent an excellent tool. The same strain may be used as an enzyme producer and non-producer, while other characteristics are identical. When genes conferring traits of interest are identified and characterized, it is possible to construct strains with desired qualities like bacteriocin production and resistance, and proteinase, peptidase and/or lipase production. So far there has been limited application of genetic techniques to industrial strains due to the lack of efficient procedures for genetic transfer. However, since the first report on transformation of Lactobacillus casei by electroporation (Chassy & Flickinger, 1987), several procedures for the electrotransformation of different Lactobacillus strains and species have been published (Aukrust & Nes, 1988; Luchansky et al., 1988, 1989; Badii et al., 1989; Josson et al., 1989; Bringel & Hubert, 1990, Gaier et al., 1990; Natori et al., 1990; Posno et al., 1991; Vescovo et al., 1991). One significant trend in the reported electroporation results has been the great strain variation in transformation efficiency, with high efficiency of transfer only for selected strains. This applies also

Homofermentative lactobacilli play a significant role in a wide range of fermented foods, and have widespread use in production and preservation of sausages and meats, vegetables and silage (Sharpe, 1981). The properties of the starter strains used play an important role for the flavour and texture of the product. It has been shown that in the production of dry sausage, the addition of lipase and proteinase before fermentation gives significant differences in off-odour, meat whiteness, bitter taste, hardness and stickiness of the sausage (Naes et al., 1991). For further evaluation of the contribution of single properties of a starter culture dur* Parts of the electroporation procedures have been presented as posters at the following meetings: (1) Third Symposium on Lactic Acid Bacteria-Genetics, Metabolism and Applications. Wageningen, The Netherlands, 17-21 September 1990. (2) Lactic-91, Lactic Acid Bacteria-Research and Industrial Applications in the Agrofood Industries. Caen, France, 12-13 September 1991. Food Research International 0963-9969/92/$05.00 0 1992 Canadian Institute of Food Science and Technology 253

T. Aukrust, H. Blom

254

to L. pluntarum, where several strains have been transformed with high efficiency. However, most strains of L. phntarum, including those with industrial potential, take up exogenous DNA at very low frequencies (Rixon et al., 1990). The present work describes the strain variations and electroporation conditions that need to be controlled to obtain efficient genetic transfer in Lactobacillus strains of meat and vegetable origin. MATERIALS AND METHODS Strains, plasmids and extraction of plasmid DNA The strains used in the study are listed in Table 1. The same preparation of plasmid pVS2, a cloning vector containing the Lactococcus luctis pSH71 replicon and the erythromycin and chloramphenico1 resistance genes from plasmids pE194 and pGB301, respectively (von Wright et al., 1987), was used in all transformation experiments. The plasmid pVS2 was isolated from L. lack using the procedure described by Anderson and McKay (1983). A TKOlOO DNA Mini-Fluorometer (Hoefer Scientific Instruments, San Francisco, CA, USA) was used to determine the DNA concentration in the plasmid preparation. Plasmid analysis of transformants was performed according to the alkaline lysis method of Maniatis et al. (1982) using 20 mg/ ml lysozyme in the lysis buffer.

Cell preparation Cells were grown in Difco Lactobacilli MRS broth with or without 1% glycine at 30°C. Logphase cultures were diluted with MRS to Al&j’ = 0.13 - 0.15 (Beckman model 25 spectrophotometer, Beckman, Fullerton, CA, USA), incubated at 30°C and harvested at A’;; = 0.6. In medium shift experiments log-phase cultures grown in MRS without dextrose were transferred to MRS with dextrose at indicated densities. When harvesting, cells were initially centrifuged at minimum g x min value and washed twice with double-distilled water, then washed and resuspended to one hundredth of initial volume in electroporation solution. Electroporation procedures Immediately before electroporation, 2 ,ul DNA solution (0.35 lg plasmid DNA) were added per 40 ~1 cell suspension. The mixture was transferred to ice-cold electroporation cuvettes and electroporated using a BioRad GenePulser with pulse control (BioRad Laboratories, Richmond, CA, USA). Unless otherwise stated, results were obtained using a constant capacitance setting of 25 PF and 2 mm electroporation cuvettes. For screening of strains the following conditions were used: (a) electroporation in 952 mM sucrose and 3.5 mM MgCl, (Luchansky et al., 1989)

Table 1. List of strains used on this study Strains L. L. L. L.

plantarum plantarum plantarum plantarum

ATCC 8014 B192 YIT 68 NC8

L. plantarum NCDO 340 L. plantarum NCDO 1193 L. plantarum NCDO 1752 L. plantarum MOP 1 L. plantarum Mtiller L. plantarum Saga II L. L. L. L. L. L.

plantarum plantarum sake sake sake sake

Ringstad 76

NCIMB 40450 b L 45 L 45 variant D Lb 706

Source Laboratory culture Laboratory culture Yakult starter strain Silage isolate Silage Ensilage of vegetables Fermented cabbage, L. plantarum type strain Mount Olive Pickle Plant Isolate from a freeze dried meat starter Isolate from a commercially available meat starter Fermented sausage startera Fermented sausage’ Fermented carrot” Fermented sausage’ Fermented sausagea Fermented sausage

“Identified by API 50 CHL. b Deposited for the purpose of patent procedure.

Obtained from American Type Culture Collection Valio Finnish Cooperative Dairies’ Association Yakult, Japan W. Dobrogosz, N. Carolina State University, Raleigh, NC, USA National Collection of Food Bacteria National Collection of Food Bacteria National Collection of Food Bacteria Mark Daeschel, Oregon State University, Corvallis, OR, USA Rudolf Mtiller & Co. Pohlheim, FRG Microlife Techniques, Florida, USA Norwegian Food Research Norwegian Food Research Norwegian Food Research Norwegian Food Research Norwegian Food Research F.-K. Liicke, Bundesanstalt Kulmbach, FRG

Institute, Aas, Norway Institute, Aas, Norway Institute, Aas, Norway Institute, Aas, Norway Institute, Aas, Norway fur Fleischforschung

Transformation of Lactobacillus strains using 100 ~1, 1.5 kV and 800 ohm; (b) electroporation in 30% (w/v) PEG 1500 (polyethylene glycol molecular weight range 1300-l 600) in doubledistilled water (Ahrnt et al., 1991) using 40 ~1, 1.5 kV and 400 ohm. To optimize the electrical conditions, varying voltages and pulse control resistances were applied. In the optimized procedure, 1% glycine was used in all growth media. For medium shift, cells grown in MRS-glycine without dextrose were diluted to A’&’ = 0.25 with MRS-glycine (with dextrose), incubated at 30°C and harvested at A’&$’= 0.6. The cells were then carefully centrifuged at minimum g X min value, washed with an equal volume 1 mM MgCl,, then with 30% PEG 1500, resuspended to one hundredth of initial volume in the same solution and electroporated as described above. After electroporation, cells were diluted 10 times in MRS with 0.5 M sucrose and 0.1 M MgCl, and incubated for 2 h at 30°C before plating on MRS agar with 5 pg/ml chloramphenicol for selection of transformants. Transformation efficiency and transformation frequency were defined as transformants/pg DNA and transformants/surviving cell, respectively. Fatty acid analysis Bacteria were grown for 48 h at 28°C on Trypticase Soy Broth Agar (TSBA), containing (w/v) 3% Trypticase Soy Broth (BBL) and 1.5% Bacto Agar (Difco). Circa 40 mg (wet weight) cells were harvested from the most dilute quadrant showing confluent growth (late log phase). Whole cell fatty acids were saponified, methylated and extracted following the procedure of Miller and Berger (1985). The MIDI microbial identification system -MIS (Microbial ID, Newark, DE, USA) was used for identification of the fatty acids. Library generating software and a statistical program CLUS developed by Microbial ID was used for the principal component and cluster analysis of strains. Response surface plots Cells from the same batch were washed with water and resuspended in 30% (w/v) PEG 1500 as described, then kept on ice. For each treatment point in the plot DNA was added to aliquots of the cell suspension, mixed, immediately transferred to the electroporation cuvette and electro-

255

porated. The voltages used were 2500, 2000, 1500 and 1000 V. At each voltage the pulse control resistance settings 100, 200, 400, 600, 800 and 1000 ohm were applied. A second degree polynomial with interactions in two variables was fitted to data using the RSREG module of the SAS system (SAS Institute Inc., Cary, NC, USA).

RESULTS Strain variations in transformation efficiency In preliminary experiments, electroporation was done in different electroporation solutions, using L. plantarum strains ATCC 8014 and NC8 for testing of the solutions and optimization of electrical conditions. The two electroporation solutions 30% PEG 1500 and 952 I’nM sucrose with 3.5 InM MgClz led to the most efficient transformation. The optimum electrical conditions in PEG 1500 were 1.5 kV with 400 ohm pulse control resistance. In sucrose-magnesium the same voltage (1.5 kV) but a higher pulse control resistance (800 ohm) to compensate for the conductivity of the solution proved most efficient. The two electroporation procedures mentioned above, without any further optimization, were used Table 2. Number of transformants/pg DNA after electroporation of different Lactobucillus strains grown in MRS using either 952 mu sucrose and 34 mu MgCl,, 1.5 kV and 800 ohm or 30% (w/v) PEG 1500, 1.5 kV and 400 obm Strain

L. plantarum ATCC 8014 L. plantarum B 192 L. plantarum YIT 68

Transformation Sucrosemagnesium

efficiency .___ 30% (w/v) PEG 1500

1 x lo? nd” nd

2 x IO4 3 x 103 nd

2x

lo4 103 10” 10” lo3 lo5

3 2 2 3

1 x lo2 nd nd nd nd nd

1 7 7 1 2 3 2

Vegetable strains L. L. L. L. L. L.

plantarum NC 8 plantarum NCDO 340 plantarum NCDO 1193 plantarum NCDO 1752 plantarum MOP 1 sake NCIMB 40450

2 1 1 5 8

x x x x x

x lo5 x 105 x lo3 x 10-7 nd 2 x 103

Meat strains L. L. L. L. L. L. L.

plantarum Miiller plantarum Ringstad plantarum Saga II plantarum 76 sake L 45 sake L45 var. D sake Lb 706

“nd = Not detectable.

x x x x x x x

104 10’ 10’ 10’ 10’ lo2 10’

256

T. Aukrust, H. Blom

to transform L. pluntarum and L. sake strains of vegetable and meat origin (Table 2). Electroporation in sucrose-magnesium was successful for all of the vegetable strains, but only for one of the meat strains tested. Electroporation in PEG 1500 resulted in successful transformation of all meat strains and five out of six vegetable strains tested. Transformation efficiencies were higher in PEG 1500 than in sucrose-magnesium, with the exception of the two vegetable strains MOP 1 and NCIMB 40450. The strain variations in transformation efficiency were pronounced, regardless of the electroporation solution used. Thus the PEG solution seemed to be best suited for use in a general transformation procedure, and was selected for further experiments. Effect of electroporation conditions described by response surface plots To study the response to varying electrical conditions, the strains were electroporated in PEG 1500 using different voltage and pulse control resistance combinations. A three-dimensional response surface plot was drawn to describe the effect on transformation efficiency. Results for strain NC8 are shown in Fig. 1A. Pulses with time constants (the time over which the voltage declines to l/e, -37% of the peak value) ranging from 2 to 20 ms

Fig. 1A. Response surface plot showing transformation efficiency of Lactobacillus plantarum NC8 as a function of voltage and pulse control using 30% (w/v) polyethylene glycol (PEG 1500) electroporation solution. The transformation efficiencies are indicated by arrows.

resulted in successful transformation. The plot has an optimum from high voltages/low pulse control resistance (short pulses) to low voltages/high pulse control resistance (long pulses), demonstrating the importance of specific combinations of voltage and pulse control resistance for efficient transformation. To evaluate the strain variation, the response of other strains were tested (Fig. 2). The main difference between strains was the level of transformation efficiencies obtained, while the pattern of the response surface plots were the same. The conditions used for screening of strains (Table 2) were located in the least variable part of the response surface when different plots were compared (Figs 1 and 2), and were optimum or close to optimum for most strains. At higher and lower voltages the strain variations were more pronounced. For further optimization of transformation efficiency for specific strains, other combinations along the optimum shown in the plots, such as 2.5 kV and 100 ohm, 2.0 kV and 100 ohm and 2.0 kV and 200 ohm, should also be tried. Selected combinations of voltage and pulse control resistance were used to confirm the response pattern of the other strains listed in Table 2. All had the type of response described, except strain 76 where field strength appeared limiting. A 5-10 times increase in transformation efficiency and a

Fig. 1B. Effect of 1% (w/v) glycine on transformation efficiency of NC8 as demonstrated by the response surface plot.

Transformation of Lactobacillus

response pattern comparable to the other strains was obtained when field strength was increased using 1 mm electroporation cuvettes (results not shown). Pretreatments used to increase transformation efficiency Even if electroporation in PEG 1500 was preferable for most of the strains used, the transformation efficiency of some strains was low. To increase transformability, 1% glycine was added to the growth medium. The response surface plot of

strains

257

strain NC8, which had the largest increase in transformation efficiency when glycine was added to the growth medium, is shown in Fig 1B. Comparison of the response surface plots of different strains grown with and without 1% glycine in the medium showed that although the transformation efficiencies were increased, the pattern of the response surface remained essentially the same. Several of the vegetable and meat strains with low transformation efficiencies in PEG 1500 produced considerable amounts of extracelluar polysaccharides. This was most pronounced in strains MOP 1, NCIMB 40450, L 45 and Lb 706. The ad-

6-

B

s-

Fig. 2.

Response surface plots showing the transformation efficiencies of selected strains as a function of voltage and pulse control using 30% (w/v) PEG 1500 electroporation solution: (A) ATCC 8014, (B) B192 and (C) NCDO 1752.

T. Aukrust, H. Blom

258

A600 at shift

no shii

medium shii

Fig. 3. Effect of medium shift and washing procedure on transformation frequency (transformantskurviving cell) of Lactobacillus sake Lb 706 with regards to density of culture at shift. ? ?Washing with water; ??washing with 1 mM MgCI, then 30% (w/v) PEG 1500.

of 1% glycine in the growth medium gave an increase in the amount of slime present after washing with water without improving the transformation efficiencies of these strains (results not shown). Lactobacihs sake L45 var. D, a variant of strain L45 without any visible slime production, had considerably higher transformation efficiency compared with the original strain (Table 2), also indicating an adverse effect of these extracellular polysaccharides. Different washing procedures were tried on the heavy slime producer L. sake Lb 706 grown in MRS with 1% glycine. The use of 1 mM MgCl, d&ion

and 30% PEG 1500 instead of water in the washing steps led to a reduction in the amount of slime present, and facilitated efficient washing and concentration of the cells. This improved the transformation results considerably (Fig. 3, no shift). However, there were still visible amounts of slime present after washing. By increasing the concentration of MgCl, (lo-100 mM) the cell suspension became easier to handle, but this also led to a reduction in pulse length and transformation efficiency at the highest voltages (results not shown). Medium shift from MRS without dextrose to MRS was performed, with shift at different densities of the culture (Fig. 3). When starved cells were used as inoculum, the amount of slime present at harvest was reduced. As seen from the results (Fig. 3), at late shift the washing procedure was less critical, but the transformation frequency was reduced when the shift was performed too close to harvest. A combination of relatively late shift (A’,$ = 0.2 - 0.3) and magnesium wash gave the best results, and was included in the procedure. The optimized procedure, including the described pre-conditioning of the cells, was used on strains with and without production of extracellular polysaccharides. This led to an increase in transformation efficiency, with the largest increase for strains producing extracellular polysaccharides in considerable amounts (Table 3). Fatty acid composition of the strains used in the experiments To evaluate whether there was any correlation between transformation efficiency, choice of pro-

Table 3. Transformation efficiency (transformantsltig DNA) of Lacfobuciflus strains with and without production of extracellular polysaccharides using the optimized procedure. Increase in transformation efficiency compared with electroporation without any preconditioning is indicated in the table Production of extracelluar polysaccharides”

Strain L. L. L. L. L. L. L. L.

plantarum plantarum plantarum plantarum sake sake sake sake

Transformation

-

efficiency

Increased by a factor of

-

1 x 105

5

NC 8

5 x 106

17

MOP 1

+

76

+

3 x 103 1 x lo2 3 x lo3 3 x 10’ 1 x 103 4 x 104

ATCC 8014

NCIMB 40450

++

U5 45 var. D

++

Lb 706

++

“Slime production was evaluated after second wash with water and centrifugation = Normal pellet. + = More than 50% supematant. f+ = Less than 50% supernatant.

at 60.500 g/min.

z 3000 10 1.5 150 3

2000

259

Transformation of Lactobacillus strains

Table 4. Fatty acid composition of the strains used in the study. The table shows results for bacteria grown on MRS with standard composition under anaerobic conditions Strain

% fatty acid C:14:0

L. plunturum ATCC 8014 L. plantarum NRRL B192 L. pluntarum YIT 68 L. L. L. L. L.

phntarum phntarum plantarum phtarum plantarum

L. L. L. L. L. L.

plantarum plantarum plantarum plantarum sake sake

C:16:1 cis9

C:16:0

C:18:1 cis9

C:18:1 cisll

C:18:0

4 4 1

5 7 2

32 26 42

8 13 9

8 12 28

2 4 3 4 4

3 7 7 6 6

40 28 30 32 18

5 8 7 10 18

7 9 5 6 8

2

NCDO 340 NCDO 1193 NCDO 1752 MOP 1 Miiller Ringstad Saga II

3 3 4

6 6 6

26 27 15

10 9 23

7 7 16 10 10 10

2

NC 8

76

6

5

21

19

L 45 L4 5 variant D

2 3

6 6

26 28

18 19

cedure and the fatty acid composition of strains from different sources, the strains used were subjected to fatty acid analysis. Results from the fatty acid analysis of bacteria grown in MRS under anaerobic conditions are shown in Table 4. Strain YIT 68, the only strain that was not transformed by any of the transformation procedures described, was clearly different from the rest of the strains having a high content of the C:16:0 and a low content of the C:19:0 cycle 9,lO fatty acid. The other strains can be divided into two distinct groups according to their content of the C: 19: 0 cycle 11,12 fatty acid also called lactobacillic acid (L. sake Lb 706 and NCIMB 40450 have not been analysed). Strains with and without lactobacillic acid were found among both vegetable and meat isolates. There were also considerable amounts of the C : 18 : 1 cis 9,lO fatty acid (oleic acid) usually connected with Carnobacterium (Collins et al., 1987) present in both groups. However, the strains were grown on MRS with standard composition. This medium contains Tween 80, the sorbitan ester of oleic acid, thus explaining the amounts of this fatty acid.

DISCUSSION

Lactobacillus plantarum ATCC 8014, one of the strains which was used for testing of electroporation solutions, has previously been electrotrans-

1 2

2

2 2

C:19:Ocyclo c9-10

C:19:Ocyclo Cl 1-12

23 38 16

20 -

19 24 23 26 44

22 20 25 15

30 30 36 38 36 35

15 18

formed with low efficiency (2 X lo2 transformants/ pg DNA) using HEPES electroporation buffer and no pulse control (Aukrust & Nes, 1988). It has also been transformed using 30% PEG 1000 as an electroporation solution with an efficiency of 5.5 X lo4 transformants/pg DNA (Bringel & Hubert, 1990). Thirty per cent PEG 1500 has been used for high efficiency transformation of L. reuteri (Ahrne et al., 1991), and was here shown to be efficient also for L. plantarum and L. sake. Using the optimized procedure, strain ATCC 8014 was transformed with higher efficiencies than those previously reported (Table 3). Several factors may contribute to the effect of PEG in electrotransformation. It is suitable for electroporation with pulse control due to the low conductivity of the solution, has been used for chemical induction of competence (Klebe et al., 1983; Sanders & Nicholson, 1987) and may protect against lysis in high current fields (Reed, 1987). Voltage or field strength, voltage and time constant, or the shape of the pulse have been reported to be the most significant factor in electroporation, with optimum values depending on species and strain (Chassy & Flickinger, 1987; Luchansky et al., 1989; Chassy et al., 1988; Hashiba et al., 1990; McIntyre & Harlander, 1989). The threedimensional plot used in this work is well suited to illustrate the combined effect of these factors. At the highest voltages, only short pulses may be used without extensive cell death resulting in a

260

T. Aukrust, H. Blom

reduced number of transformants. At lower voltages, longer pulses are necessary to obtain the desired effect on the cells. The response surface plots illustrate how the pulse length should be adjusted at different voltages to obtain efficient electropermeation of DNA. The plots shown are obtained using an electroporation solution with low conductivity, hence the resistance set by the pulse control of the apparatus is the major contributor to the length of the pulse. When more conductive electroporation solutions are used, the pulse control should be adjusted accordingly, as indicated by our results with sucrosemagnesium. It is not clear how DNA traverses the murein layer, and why the cell wall appears to be a barrier in some cases and not in others. Additions to the growth medium like threonine, glycine and sorbitol have been shown to increase the transformation efficiency of Lactobacillus species, but the effect is strain variable (Aukrust & Nes, 1988; Bringel & Hubert, 1990; Hashiba et al., 1990; Posno et al., 1991). The addition of glycine in the growth medium inhibits formation of cross-linkages in the cell wall where L-alanine is replaced by glycine (Hammes et al., 1973), thereby weakening the cell wall. This might also affect the response to electric pulse. According to the response surface plots for strains grown with and without glycine (Fig. 1) the positive effect of glycine is due to a general increase in electropermeation of DNA, while the pulse characteristics for efficient transformation remains mainly unchanged. The similarity between the response surfaces of different strains with and without pre-conditioning of the cells (Figs 1 and 2), simplifies transformation of new strains by electroporation. A standard setting of the apparatus may be used for screening of strains for evaluation of transformability, and the use of a few selected combinations of voltage plus pulse control along the ridge of optimum transformation is sufficient for control of response pattern and optimization efficiency of specific strains. Our results also show that extracelluar polysaccharides have an adverse effect on transformation efficiency. They may represent an additional barrier to DNA penetration, or, more likely, the effect may be due to less efficient washing and concentration of the cells. When magnesium was included in the washing solution there was less dissociated slime present after washing, giving a positive effect on transformation frequency. When medium shift

was included in the procedure to reduce slime production, this had an additional effect on the transformation results (Fig. 3). The combined effect of these pre-treatments increased the transformation frequency of this strain by a factor of lo3 using 1% (w/v) glycine in the growth medium. Little is known regarding the physical chemistry of a bacterial membrane during a high voltage pulse, and it is unknown what influence membrane composition has on electrophoretic success (Mercenier & Chassy, 1988). The strains used, except strain YIT 68, had similar fatty acid profiles and could be divided in two distinct groups according to their content of lactobacillic acid. Strain YIT 68, which was different from the rest of the strains, was not transformed by either of the procedures tried. All strains which contained lactobacillic acid were transformed with reasonable efficiency, while the group lacking this fatty acid exhibited lower transformation efficiencies and more varied results. There was no direct correlation between the fatty acid composition and the origin of the strains, although in the vegetable group, which was easiest to transform, more strains contained lactobacillic acid. Our results indicate that important properties for electro-transformation may not only be species specific, but also reflect the origin of the strain. Although the described PEG procedure worked well for most strains, and had to be used for successful transformation of the meat strains in the study, it was not optimal for all strains. All vegetable strains were transformed using sucrosemagnesium, and the vegetable strains MOP 1 and NCIMB 40450 had higher transformation efficiencies using this electroporation solution. Thus, for proper choice of electroporation procedure, the source of the strain should be considered. The electroporation conditions described in this paper have proved useful also for species other than L. plantarum and L. sake. Previously untransformable Lactobacillus casei and Streptococcus salivarius subsp. thermophilus strains have been subjected to the PEG 1500 procedure, obtaining 8 X lo5 and lo6 transformants/pg DNA, respectively (Anna-Maija Taimisto, Valio, Helsinki, Finland, pers. comm., 1991). Thus the procedures and approaches to electrotransformation described in this paper may be an important tool for the increase of transformation efficiencies to a level sufficient for direct shot gun cloning not only for L. plantarum and L. sake, which was used in this study, but also for other strains used for food production.

Transformation of Lactobacillus strains

ACKNOWLEDGEMENTS This work was partially financed by the Nordic Industrial Fund. We thank Professor SalkinojaSalonen, University of Helsinki, for performing the fatty acid analysis and Tormod Naes, MATFORSK, for the data presentation of the electroporation results. We also thank Anna-Maija Taimisto for helpful discussions and Brit Oppegaard Pedersen for skilful technical assistance.

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