BIOTECHNOLOGY TECHNIQUES Voltme 20 No. 8 (August 1996) pp.553-558 Received as revised 20 May

Optimized Transformation by Electroporation of Lactococcus lactis IL1 403

Woo-Jin Kim, Tae-Ryeon Heo and Jae-Seong So* Department of Biological Engineering, Inha University, 253 YongHyun-Dong,

Nam-Gu, Inchon 402-751, Korea

Summary

Rolling circle replication of plasmid pGKV21 in L. luctis was a more stable

replication mode than the theta replication of plasmid pIL253. When the plasmid pGKV21 was used to develop and optimize transformation of L. fsctis by electroporation, optimum transformation was obtained using dense suspension of late-exponential phase cells subjected to a high voltage (12.5 kV/cm) and a short pulse time (0.5 ms) in the presence of a plasmid DNA. With plasmid vector pGKV21, a transformation frequency

of 2.2 X lo8 transformants per fig of DNA was obtained.

Introduction

Luctococcus Iuctis is an important starter organism in cheese production. Interests in its genetic manipulation have mainly focused on improving its ability for lactose fermentation, casein breakdown, and resistance against the attack by bacteriophage (Lucy et al., 1993). Efficient DNA transformation system is a prerequisite for the application of recombinant DNA technology to L. luctis and the efficient DNA transformation requires the right plasmid to be used (Kiewiet et al., 1993). In this study the effect of plasmid stability in L. fuctis was studied with plasmids of two replication modes: the rolling circle(pGKV21) and theta modes(pIL252, pIL253). As a result, the rolling circle replication plasmid pGKV21 is more stable than the theta replication pIL253. Subsequently, the plasmid pGKV21 was used to develop and optimize transformation of L. /u&s by electroporation. Transformation by electroporation involves the application of high-voltage electric field pulses of short duration to induce the formation of transient pores in the membranes of cells. This technique has been proved useful in bacterial species previously regarded as untransformable. McIntyre and Harlander(l989) transformed L. factis subsp. Iuctis by electroporation only with low transformation frequency. Subsequently Ho10 and Nes(1989) transformed several strains of L. luctis and L. crenzoris by electroporation. The highest transformation frequency obtained was 1.4 X 10’ transformants per pg of pIL253 DNA in L. fuctis subsp. luctis LM2336. However, the plasmid pIL253 DNA was found to be unstable. Although plasmid plL253, derived from a multicopy PAM B l-derivative, is a high copy number plasmid, plasmid pIL253 has disadvantages that the presence of insert DNA can not be judged on the basis of a simple test such as markei inactivation and that it lacks the stability determinant (D’Angio et al., 1994). In this study, we have attempted to improve transformation frequency of L. (actis by usin g a different kind of plasmid. The plasmid pGKV21 is a vector derived from the Iactococcal plasmid pWVO1, which can be used for the isolation of promoter and transcriptional termination signals in lactic acid

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bacteria. In addition, plasmid pGKV21 has been shown to replicate in both Gram- positive and Gram-negative bacteria. Our attempt to use plasmid pGKV21 in transforming L. hctis IL1403 was successful, and the highest transformation frequency of 2.2 X lo8 transformants per ~1s DNA was obtained.

Materials and Methods

Bacterial strains and plasmid

1,. ktis IL1403 (plasmid free, restriction/modification minus) was kindly provided by A. Chopin, lnstitut National de la Recherche Agronomique, Jouy-en-Josas, France(Simon and Chopin, 1988). E. co/i TGI (pGKV21) was kindly provided by J. D. Bahk, Department of Biochemistry, Gyeongsang National University, Chinju 660-701, Korea(Jeong et al., 1993).

Isolation of plasmid DNA

Plasmid DNA was isolated from E. coli TGI by the alkaline lysis procedure. Purification of plasmid DNA by CsCl density gradient centrifugation in the presence of ethidium bromide and analysis by agarose gel electrophoresis were done by standard methods.

Determination of plasmid stability,

Single colonies of piasmid-carrying strains, which had been puriiied by two successive platings, were used to inoculate Ml7 medium containing erythromycin(5hg/tQ’). At an

optical density at 600 ,uo of 0.5, the culture was diluted IO”-fo Id in 100 I& of prewarmed Ml7 medium without erythromycin. The cells were grown for about 100 - 200 generations in successive batch cultures so that the optical density remained under 1.0 at 600 ,,,,,. This prevented the cells from entering the stationary phase of growth. At regular intervals, appropriate dilutions of the cultures were plated on nonselective Ml7 (with lactose) agar. At least 200 individual colonies from each sample were transferred to plates containing erythromycin. Resistance to the antibiotic was correlated with the presence of a plasmid. The stability of plasmids pIL252, pIL253 and pGKV21 was examined in terms of the copy number and replication mode. Results were obtained by plotting the logarithm of the percentage of plasmid-bearing cells versus the number of generations.

DNA transformation by electroporation

An overnight culture of I.. /actis IL1403 (2 &) was inoculated into 100 III! modified MRS broth and grown to various growth phases Ioptical density at 600 nm : 0.1 (1.5 X

10 cfulm!), 0.5 (1.8X lOa cfu/&), 0.95 (1.2X 10” cfu/ti), 1.5 (2.4X 10” cfu/&) and 1.9 (3.1 X 10’” cfu/m!) respectively]. Cells were harvested by centrifugation (5,000 x g, 10 min, 4°C) and washed twice with an equal volume of ice-cold electroporation buffer containing I mM HEPES (pH 7.0) and 0.5 M sucrose, resuspended in l/150 culture volume of electroporation buffer and then stored in aliquots at -70 “C until use. The cell suspensions were thawed on ice. Portions (6.4 @) were mixed with 1 $ of pGKV21

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DNA dissolved in 10 mM TrislHCL-1 mM EDTA (pH 8.0) and then transferred into the PG240-Bacterial Electrode pins (0.3 mm gap) and electroporated immediately using a single electric pulse. The pulse was delivered by the Progenitor 11 (Hoefer Sci., California,

USA) set at 100 FF and at 12.5 kV/cm. Immediately following the discharge, the

suspensions were mixed with 0.99 a~/ of ice-cold modified MRS and left on ice for 5 min. Appropriate dilutions were made in modified MRS and the cells were incubated at 30 “C for 2 h, then loo-$ portions were spread on modified MRS agar plates containing 5 pg of erythromycin per m!. Transformants appeared after 24 h of incubation at 30 “C. Actual DNA transfer was confirmed by checking the presence of plasmid DNA by agarose gel electrophoresis (O’Sullivan and Klaenhammer, 1993).

Results and Discussion

Plasmid pGKV21, which replicates as a rolling circle, was more stable than the theta replication plasmids pIL252, pIL253(Fig. I). As a result, the plasmid pGKV21 was used to develop and optimize transformation frequency of /,. /actis IL1403. L. lnctis IL1403 was successfully electroporated to a frequency of 10’ transformants with a plasmid pCKV21 (Fig. 2). No colonies were observed on control plates (omission of plasmid, pulse, or both). The highest transformation efficiency of 2.2 X 10s transformants/pg DNA was obtained with field strength of 12.5 kV/cm, pulse time of 0.5 ms, cell growth phase of 01)600,.=0. 95, and DNA concentration of 120 rig/d. The effects of several parameters on transformation frequency were systematically examined and the results are summarized as follows. The data are presented with standard deviation calculated on triplicate values obtained from at least two independent experiments.

Plasmid stability

The specific growth rate( j..t= 0.57/h) of p&mid-free L. hctis IL1403 was the highest of all the L. hctis. However, the

specific growth rate (l..t ) of L. factis IL1835(pIL252) as 0.50/h, the specific growth rate (f-t) of I.. hctis IL1837(pIL253) was 0.52/h and the specific growth rate (cl) of L. luctis IL1019(pGKV21) was 0.54/h. It appeared that the transformed cell had so much burden of metabolic pathway that it grew slowly in comparison with the plasmid- free cell. After determining the generation time of each L. luctis, the stability of plasmid pIL252, pIL253 and pGKV21 was examined after every 10-20 generations (Fig. 1). Results were obtained by plotting the logarithm of the

10

l plL253 A . plL253

* . A pGKV21

A

. *

‘:

0 50 100 150 200

Generations

Fig. I. Kinetics of plasmid stability in L. lactis.

percentage of plasmid-bearing cells versus the number of generations. Plasmid pIL252 was unstable, since it remained in only 10% of the cells after 60 generations. In contrast to pIL252, the loss of about 90% of plasmid pIL253 and 80% of plasmid pGKV21 occurred after 180 or 200 generations, respectively. Therefore, plasmid pGKV2I was

.

/ I

4 6 8 10 12 14 16 18

Field Strength (kV/cm)

Fig. 2. Effect of plasmid concentration on transformation efficiency.

Fig. 3. Effect of field strength on transformation efficiency.

most stable of the three plasmids. In comparing with the copy number of pIasmid(the same replication mode), the high copy of plasmid pIL253 was much more stable than the low copy of plasmid pIL252. Furthermore, in comparison with the replication mode of plasmid, the rolling circle replication mode of plasmid pGKV21 was more stable than the theta replication mode of plasmid pIL253. This is in contrast to the previous report on the plasmid stability of L. In&s. Kiewiet et aL(1993) reported that there was no difference in plasmid stability between rolling circle replication plasmid (pKS100) and theta replication plasmid (pAMS100). Interestingly, the longer the size of the theta replication plasmid [ pIL252 (4.6kb) < pIL253(4.8kb) < pAMSlOO(8.4kb) ], the more stable. However, the shorter the size of the rolling circle replication plasmid [pSK105L(lOkb) < pKS100(5kb)], the more stable. Taken together, it appears that the plasmid stability in lactococci is affected by replication origin and size in addition to replication mode.

Effect of plasmid concentration on transformation efficiency

DNA concentration did not significantly affect on transformation efficiency, since the actual number of transformants linearly increased up from 0.1 ng/ti to 100 ng/ti DNA

used. However, the transformation efficiency decreased nearly lo-fold between 100 ng/& and 1000 ng/oQ’ and no transformants were detected at 0.01 ng/& (Fig. 2). When the efficiency was calculated by dividing the actual number of transformants by the amount of DNA used. The highest transformation efficiency obtained was 2.2 x lo*

transformants/pg DNA. This is approximately 15 times higher than previously reported

efficiency of 1.4 X 10’ transformants/@ DNA (Ho10 and Nes, 1989).

Effect of field strength on transformation efficiency

Field strengths of 4, 6.25, 8, 12.5, and 20 kV/cm were generated using 100 FF capacitor with a 0.3 mm electrode gap. Pulse time was set at 0.5 ms. As shown in Fig. 3, the transformation efficiency increased sharply with field strengths increasing from 4 to 12.5 kV/cm and then decreased rapidly with field strengths from 12.5 to 20 kV/cm. Maximum transformation efficiency was observed at a field strength of 12.5 kVlcm.

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1 2 3 4 5 2

Pulse time ( ms ) Cell Concentration ( O.D. )

Fig. 4. Effect of pulse time on Fig. 5. Effect of culture age on transformation efficiency. transformation efficiency

Effect of pulse length on transformation efficiency

Pulse length was increased from 0.1 to 5 ms with field strength set at 12.5 kV/cm. Increasing the pulse length from 0.1 to 0.5 ms resulted in a lOO-fold increase in transformation efficiency, while an increase from 0.5 to 5 ms resulted in approximately 60-fold drop in transformation efficiency (Fig. 4). This is in contrast to the result obtained with L. /actis (Holo and Nes, 1989), where the maximum efficiency was obtained at 5 ms. There could be several explanations for this difference, including the different kind of electroporation apparatus, plasmid DNA, electroporation buffer, and sample volume.

Effect of growth phase on transformation efficiency

Samples were harvested by centrifugation at O.D.C~~,~ = 0.1, 0.5, 0.95, 1.5, 1.90. Maximum efficiency of 2.2 X lo8 transformants/pg DNA was observed at late-exponential

phase (ODWXI~,,, = 0.95) (Fig. 5). This differs from results obtained with L. luctis (Holo and Nes, 1989) and L. kelviticus (Bhowmik and Steele, 1993), in which the highest transformation frequency was obtained in the early-exponential or mid-exponential phase.

Effect of the buffer on transformation efficiency

Cells were grown to OD600,,n, = 1.0, harvested by centrifugation, washed with various buffers, and electroporated using the optimal parameters described above. As shown in Table l., use of 1 mM HEPES buffer (pH 7.0) containing 0.5 M sucrose, as the osmotic stabilizer and cryoprotectant, gave the highest transformation efficiency. Interestingly, the increase of HEPES concentration negatively affected electroporation of L. lactis. This result shows that the increasing conductivity of buffer solution may negatively affect the transformation efficiency.

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Buffet % Transformation efficiency Sterile water 6.5

0.5 M sucrose 2.5 10% Glycerol 33.5

1 mM HEPES (pH 7.0) 100 5 mM HEPES (pH 7.0) 52.5

20 mM HEPES (pH 7.0) 21.4

Table I. Effect of buffer on transformation efficiency. Highest value obtained was set 100% and others were normalized against it.

In summary, this study demonstrates that electroporation is a simple, reliable, and efficient method for the transfer of plasmid DNA into lactic acid bacteria. Although there have been previous reports on electrotransformation of L. ludis (McIntyre and Harlander, 1989; Holo and Nes, 1989), by improving several parameters, we have obtained routine transformation efficiency as high as 108/pg DNA. This procedure will have broad application for genetic manipulation of many industrially important microorganisms, recalcitrant to current gene transfer methods, including lactic acid bacteria.

Acknowledgements This research was supported by grants from KOSEF and Inha [Jniversity.

References Bhowmik T. and J. L. Steele (1993), J. Gen. Microbial., 139, 1433-1439. D’Angio, C., C. B al, C. Y. Boquien, P. Langella and G. Corrieu (1994), FEMS Microbial. Lett., 116, 25-30. Holo H. and 1. F. Nes (1989), Appl. Environ. Microbial., 55, 3119-3123. Jeong, J. Y., H. S. Seo, H. Y. Kim, C. K. Choi, M. J. Cho and J. D. Bahlt (I 993), Mol. Cells, 3, 373-378. Kiewiet R.., J. Kok, J. F. M. L. Seegers, G. Venema and S. Bron (1993), Appl. Environ. Microbial., 59, 358-364. Lucy M., C. Daly and G. Fitzgerald (1993), FEMS Microbial. Lett.,llO, 249-256. McIntyre D. A. and S. K. Hal-lander (1989), Appl. Environ. Microbial., 55, 604-610. O’Sullivan, D. J. and T. R, Klaenhammer (1993) Appl. Environ. Microbial., 59, 2730-2733. Simon, D. and A. Chopin (198X), Biochimie 70, 559-566.

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