APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1999, p. 3800–3804 0099-2240/99/$04.00⫹0 Copyright © 1999, American Society for Microbiology. All Rights Reserved.
Vol. 65, No. 9
An Effective Strategy, Applicable to Streptococcus salivarius and Related Bacteria, To Enhance or Confer Electroporation Competence NICOLE D. BUCKLEY,1 CHRISTIAN VADEBONCOEUR,1 DONALD J. LEBLANC,2† LINDA N. LEE,3 1 AND MICHEL FRENETTE * ´ cologie Buccale, De´partement de Biochimie et de Microbiologie, Faculte´ de Sciences et de Groupe de Recherche en E Ge´nie, et Faculte´ de Me´decine Dentaire, Universite´ Laval, Que´bec, Canada, G1K 1P41; Department of Oral Biology, Indiana University School of Dentistry, Indianapolis, Indiana 462022; and Department of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284-77583 Received 2 March 1999/Accepted 10 June 1999
Despite the large number of techniques available for transformation of bacteria, certain species and strains are still resistant to introduction of foreign DNA. Some oral streptococci are among the organisms that can be particularly difficult to transform. We performed a series of experiments that involved manipulation of growth and recovery media and cell wall weakening, in which the electroporation conditions, cell concentration, and type and concentration of the transforming plasmid were varied. The variables were optimized such that a previously untransformable Streptococcus salivarius strain, ATCC 25975, could be transformed reproducibly at a level of 105 transformants per g of DNA. The technique was used to introduce a plasmid into other strains of S. salivarius, including a fresh isolate. Moreover, the same technique was applied successfully to a wide range of oral streptococci and other gram-positive bacteria. petence, what Saunders et al. (22) called chemical competence would have to be induced. Thus, transformation techniques for members of nonnaturally competent streptococci have been developed. Transformation of Streptococcus bovis involves generation of protoplasts, followed by transformation (13). Conjugation (or mobilization by a conjugative plasmid) is a reproducible, efficient method for introducing DNA into S. sobrinus (1a), and electroporation of whole cells has been used with several different Streptococcus species and strains (2, 4, 10, 12, 13, 19, 23, 24, 27). The different techniques are needed because what works for one strain or species does not necessarily work for another. In our laboratory, numerous attempts were made over several years to transform S. salivarius ATCC 25975. All were unsuccessful. In this paper we describe a straightforward, reproducible method for producing high-efficiency, electroporation-competent S. salivarius. Our protocol involves glycine treatment of cells growing in rich medium, followed by electroporation. The technique was used successfully with other streptococci and related bacteria. (Some of the results were presented at the 75th General Session and Exhibition of the International Association for Dental Research, Orlando, Fla., 1997.)
Genetic manipulation of bacteria has become a powerful tool for elucidating fundamental biological mechanisms. While there is a plethora of techniques available to introduce foreign DNA into several bacterial species (for reviews see references 15 and 22), some bacteria have proved to be refractory to most or all of the protocols that have been described. Occasionally, there is an entire species whose members are difficult to transform, such as Streptococcus salivarius or Streptococcus sobrinus, while in other species (e.g., Streptococcus mutans) there are large differences in transformability among strains. Characterization of such species is difficult, as it relies on often painstaking, alternative approaches. Moreover, in some cases, information accumulated with one bacterial strain cannot be built upon, and the strain is abandoned in favor of a related, more amenable strain. Certain streptococci can become naturally competent; i.e., they can take up free DNA from the surrounding medium (6). Natural competence is expressed by these bacteria during growth under defined conditions, usually only at certain stages of growth (22). The streptococci have been divided into the following six major phylogenetic clusters based on their 16S RNA sequences: the pyogenic group, the anginosus group, the mitis group, the mutans group, the bovis group, and the salivarius group (9). Natural competence of streptococci has been associated with the products of a few genes, and in a survey, Håverstein et al. (6) found that these genes were present in strains belonging to the mitis and anginosus groups and more rarely in members of the mutans group. Strains belonging to the other groups lacked these genes, as well as the associated natural competence phenotype. In the absence of natural com-
MATERIALS AND METHODS Bacterial strains, plasmids, and media. The electroporation technique described in this paper was developed and optimized for S. salivarius ATCC 25975 (kindly provided by I. R. Hamilton, University of Manitoba, Winnipeg, Manitoba, Canada). The following other bacteria were also subjected to the electroporation technique described here: Lactococcus lactis LM 0230 (16), Pediococcus acidilactici SMQ-250, Streptococcus thermophilus DT1 (now designated S. thermophilus SMQ-310), all of which were generously provided by S. Moineau, Laval University, Quebec, Canada; S. salivarius ATCC 7073 and ATCC 13419 and a fresh S. salivarius isolate, strain 30.1 (18); S. sobrinus 6715 (26); Streptococcus vestibularis ATCC 49124; S. thermophilus ATCC 19258; Streptococcus sanguis ATCC 10556; and S. mutans XS123 (30). An Escherichia coli-Streptococcus shuttle vector, pDL278 (11), and a bridge vector, pNZ123 (3), were used in this study. These vectors were maintained in E. coli XL1 Blue (Stratagene, La Jolla, Calif.) grown at 37°C with agitation in Luria-Bertani medium (21) supplemented with 100 g of spectinomycin per ml and 10 g of chloramphenicol per ml, respec-
* Corresponding author. Mailing address: Groupe de Recherche en ´ cologie Buccale, Faculte´ de Me´decine Dentaire, Universite´ Laval, E Que´bec, Canada G1K 7P4. Phone: (418) 656-2131, ext. 5502. Fax: (418) 656-2861. E-mail:
[email protected]. † Present address: Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285. 3800
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tively. These antibiotics were used at the same concentrations when we selected for the plasmids in streptococci and related bacteria. Tryptone-yeast extractglucose broth (TYE) (18), Hogg-Jago glucose broth (HJG) (12), and M17 broth (28) were used for cultivation of streptococci. Medium designations to which an S has been added indicate the addition of sorbitol to a final concentration of 0.4 M. Streptococci and related bacteria were incubated aerobically at 37°C without agitation. Agar was added to a final concentration of 1.5% when agar petri plates were prepared. Chemicals were purchased from Sigma Chemical Co. (St. Louis, Mo.), while Difco Laboratories (Detroit, Mich.) supplied other medium reagents. Culture purity was verified by microscopic examination of cells, by determining the homogeneity of isolated colonies grown on nonselective media, and by performing carbohydrate fermentation tests. Cell preparation and electroporation. Culture growth was monitored by measuring the optical density at 660 nm (OD660) with a Spectronic 20 spectrophotometer (Milton Roy Co., Rochester, N.Y.). Glycine was added to cultures at the concentrations and times indicated below, and cells were collected by centrifugation, washed twice in electroporation buffer (EPB) (5 mM potassium phosphate with or without MgCl2 and 10% glycerol), frozen in an ethanol dry ice bath, and stored at ⫺80°C. For electroporation, cells were thawed on ice, combined with 200 ng to 1 g of pNZ123 or 1 g of pDL278, and transferred to prechilled 1-mm electroporation cuvettes (Bio-Rad Laboratories, Richmond, Calif.). Electroporation was performed with a Bio-Rad Gene Pulser apparatus set at 25 F with an attached Bio-Rad pulse controller that allowed us to adjust the resistance. Transformation analysis. To optimize the transformation process, multiple variables were examined in single experiments by using the multifactorial experimental designs and analyses described by Marciset and Mollet (12). Factors were tested at maximal, intermediate, and minimal levels (indicated in designations by ⫹, 0, and ⫺, respectively). Trial runs with all of the test factors at intermediate levels were performed in triplicate or quadruplicate, and the variation coefficient [VC(0)] was determined as follows: VC(0) ⫽ n ⫺ 1/n ⫻ 100, where n is ¥ni/n (the average number of transformants obtained under intermediate conditions) and n⫺1 is the standard variation, {¥[(ni ⫺ n )2])/(n ⫺ 1)}. Test runs in which factors were at the maximal or minimal levels were analyzed as follows. The variation coefficient (VC) for an individual test factor (F1) was determined as follows: VC(F1) ⫽ 100/a ⫻ [¥(F1 ⫹ runs) ⫺ ¥(F1 ⫺ runs)]/8, where a is ¥ai/8 (the average number of transformants obtained for eight tests runs). The interaction between two factors (F1 and F2) or the variation coefficient [VC(F1,F2)] was determined as follows: VC(F1,F2) ⫽ 100/a ⫻ [(¥F1,F2 ⫹,⫹ runs and ⫺,⫺) ⫺ (¥F1,F2 ⫺,⫹ runs and ⫹,⫺)]/8. The absolute values of VC(F1) were compared with the absolute value of VC(0), and differences greater than zero were considered possibly significant and differences more than twice VC(0) were considered very significant. Marciset and Mollet (12) stated that the optimal level of a factor was when its variation coefficient changed sign after its maximal value increased or its minimal value decreased from one experiment to the next. Plasmid DNA isolation. Plasmid DNA was isolated from E. coli by the alkaline lysis method described by Sambrook et al. (21), and large-batch preparations were obtained with a Qiagen Plasmid Maxi kit (Qiagen Inc., Chatsworth, Calif.). Plasmid DNA were isolated from streptococci and related bacteria by using the method of O’Sullivan and Klaenhammer (17), with the following modifications: ethidium bromide was omitted from the preparations, mutanolysin (12 U/ml) was added to the lysozyme solution used for oral streptococci, and the incubation time in the presence of lysozyme-mutanolysin was increased from 15 min to 1 h. Plasmid DNA was quantified by agarose gel electrophoresis followed by staining with ethidium bromide and comparison to an EcoRI-HindIII-digested standard (Pharmacia Biotech Inc., Baie d’Urfe, Canada). Southern blot hybridization. Restriction endonuclease-digested plasmid DNA was separated by agarose gel electrophoresis, and the DNA fragments were transferred from the gels to Hybond-N nylon membranes (Amersham Life Science, Buckinghamshire, England) by using a Posiblot pressure blotter (Stratagene). The blots were then treated and hybridized with labelled plasmid DNA probes by using a nonradioactive DNA labeling and detection kit (Roche Diagnostics, Laval, Canada). EcoRI-HindIII-digested (Pharmacia Biotech Inc.) was included in all hybridization experiments as a molecular weight marker. Plasmid stability. The stability of pDL278 and pNZ123 in S. salivarius grown in HJG was tested as previously described (1a). Briefly, cultures were grown overnight and then serially diluted with fresh HJG containing 0.4 M (final concentration) sorbitol (HJGS) with or without antibiotic and incubated at 37°C. The cultures were incubated until they reached an OD660 of 0.5. Dilutions of the cultures were spread onto HJG agar plates without antibiotic, which then were incubated for 24 to 48 h. Two hundred isolated colonies were then picked onto fresh plates with and without antibiotic and incubated for 48 h. The isolates were scored for the presence of plasmids on the basis of their resistance to the appropriate antibiotics.
RESULTS AND DISCUSSION Optimization of S. salivarius transformation. It has not been possible to transform S. salivarius ATCC 25975 by previously
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described protocols that are effective with other oral streptococci and related bacteria. These protocols include electroporation of cells grown in the presence of 40 mM DL-threonine, as described by Suvorov et al. (27), and a wide variety of other electroporation protocols (4, 14, 19, 23, 24). In addition, mobilization, as described by Buckley et al. (1a), did not yield transconjugants of S. salivarius ATCC 25975. The flow chart for “devising a plasmid transformation protocol in a ‘new’ bacterial species,” as described by Saunders et al. (22), had been followed to the box labelled “give up.” Glycine is commonly used as a cell wall-weakening agent prior to electroporation (2–4, 7, 8, 25, 27). The cell wall of S. salivarius should be susceptible to glycine because its interpeptide bridges contain both L- and D-alanine (20), which are replaced by glycine, which, in turn, impedes synthesis and assembly of the cell wall (5, 29). Thus, we attempted to produce electroporation-competent S. salivarius by growing cells with glycine. A single transformant of S. salivarius ATCC 25975 was obtained after electroporation of cells grown in TYE containing 1.5% glycine. However, this successful transformation was not reproducible when we used pNZ123 isolated from either S. salivarius or E. coli. One major problem was the inconsistency of growth in the presence of glycine. Dunny et al. (4) previously observed that the glycine concentration that allowed growth of cells (albeit slowly) in one experiment resulted in complete growth inhibition in the next experiment. We predicted that addition of glycine to an exponentially growing culture would maximize the deleterious effects of this compound on cell walls, whose synthesis must occur rapidly, and at the same time ensure that there is a sufficient number of cells for preparation of competent cells. The only other study that we are aware of in which glycine was added to late-stage cultures is a study in which cryotransformable Bacillus anthracis was prepared (25). Stepanov et al. (25) grew cells to the mid-log phase in the presence of 5% glycine prior to freezing in the presence of a plasmid and then thawed the cells and screened for transformants. Thus, to overcome glycine toxicity, glycine was added to cultures during early exponential growth. In addition, rich medium was substituted for TYE, and 0.4 M sorbitol, a sugar not metabolized by S. salivarius, was added to growth medium and EPB as an osmoprotectant. A comparison of the efficiencies of transformation of pNZ123 into cells grown in three different media indicated that more transformants (1,351 versus 0 to 5 transformants/g) were obtained with cells grown in HJGS than with cells grown in either MRSS or M17S. Thus, HJGS was the medium used for all subsequent experiments. The multifactorial experimental design and statistical analyses described by Marciset and Mollet (12) were used to optimize transformation efficiency. A strong point of the method of Marciset and Mollet is that it allows interactions between factors to be identified. Intuitively, a researcher may or may not appreciate interactions, but Marciset and Mollet (12) describe a technique for rapidly and easily quantitating these relationships. The results of an experiment in which glycine concentration, the optical density when glycine was added, the concentration factor of the cells (initial cell volume/final cell volume), MgCl2 content, and the pH of EPB were examined are shown in Table 1. The sevenfold difference in the numbers of transformants obtained in the trial and test runs (n ⫽ 3.9 ⫻ 104 transformants [Tf]/g versus a ⫽ 5.6 ⫻ 103 Tf/g) indicates that there were strong interactions between factors. The VC(0) value was 22%, while in the test runs VC(glycine) was ⫺73%, which suggested that the glycine concentration tested was too high. VC(OD) was 10%, which suggested that the optical densities used in the trials were too low. VC(CF) was ⫺14%, which
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TABLE 1. Effects of glycine concentration, cell density when glycine is added, concentration factor of cells, pH, and MgCl2 concentration in EPB on the electrotransformation efficiency of S. salivarius ATCC 25975 with pNZ123a Glycine concnb
OD660c
Cell concn factord
pHe
MgCl2 concnf
Transformants/g
⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫹ 0 0 0 0
⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ 0 0 0 0
⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ 0 0 0 0
⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ 0 0 0 0
⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ 0 0 0 0
6.50 ⫻ 102 1.10 ⫻ 103 3.80 ⫻ 103 1.45 ⫻ 104 2.83 ⫻ 103 4.50 ⫻ 102 2.01 ⫻ 104 1.20 ⫻ 103 5.09 ⫻ 104 3.90 ⫻ 104 3.70 ⫻ 104 3.00 ⫻ 104
a Cells were grown in HJGS. Glycine was added to cultures at different OD660, and the cultures were incubated for an additional 2 h. b ⫺, 5% glycine; 0, 10% glycine; ⫹, 15% glycine. c ⫺, OD660 ⫽ 0.3; 0, OD660 ⫽ 0.4; ⫹, OD660 ⫽ 0.5. d ⫺, 22.5; 0, 40; ⫹, 60. e ⫺, pH 4.7; 0, pH 5.5; ⫹, pH 6.0. f ⫺, no MgCl2; 0, 2 mM MgCl2; ⫹, 4 mM MgCl2.
led to trials at higher values. VC (pH) was 3%, which prompted a decrease in the pH of the EPB. Modifications of variables that were suggested by the results of the experiment described above were tested in a second experiment (Table 2). In this case, there was little difference between n (3.0 ⫻ 104 Tf/g) and a (2.9 ⫻ 104 Tf/g) which suggested that the factors were within optimal levels. VC(0) was 33%, while VC(glycine) was 70%. The change in the sign of the VC value from the previous experiment suggested that the overlapping values were optimal; therefore, 10% glycine was used. VC(OD) was reduced to 9%, suggesting that the optical densities were within an acceptable range. Therefore, in subsequent experiments, glycine was added to cultures when the OD660 of the culture was 0.5. VC(pH) was ⫺44%, which TABLE 2. Effects of glycine concentration, cell density when glycine is added, concentration factor of cells, and MgCl2 concentration in EPB on the transformation efficiency of S. salivarius ATCC 25975 with pNZ123a Glycine concnb
OD660c
Cell concn factord
pHe
MgCl2 concnf
Transformants/g
⫹ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫹ 0 0 0
⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ 0 0 0
⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ 0 0 0
⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ 0 0 0
⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ 0 0 0
1.78 ⫻ 104 1.03 ⫻ 104 6.29 ⫻ 104 Lg 2.00 ⫻ 103 7.95 ⫻ 104 1.83 ⫻ 104 9.25 ⫻ 103 3.99 ⫻ 104 3.00 ⫻ 104 2.01 ⫻ 104
a Cells were grown in HJGS. Glycine was added to cultures at different OD660, and the cultures were incubated for an additional 2 h. b ⫺, 2.5% glycine; 0, 5% glycine; ⫹, 10% glycine. c ⫺, OD660 ⫽ 0.4; 0, OD660 ⫽ 0.6; ⫹, OD660 ⫽ 0.8. d ⫺, 40; 0, 60; ⫹, 80. e ⫺, pH 4.5; 0, pH 5.0; ⫹, pH 5.5. f ⫺, no MgCl2; 0, 2 mM MgCl2; ⫹, 4 mM MgCl2. g L, the culture was lost.
TABLE 3. Effects of electroporation parameters, length of incubation in the presence of 10% glycine, and pH of EPB on the transformation efficiency of S. salivarius ATCC 25975a Resistanceb
Voltagec
Length of incubation with glycined
pHe
Transformants/g
⫺ ⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ 0 0 0
⫺ ⫹ ⫺ ⫹ ⫺ ⫹ ⫺ ⫹ 0 0 0
⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫹ ⫹ 0 0 0
⫺ ⫹ ⫹ ⫺ ⫹ ⫺ ⫺ ⫹ 0 0 0
7.00 ⫻ 103 1.23 ⫻ 105 1.32 ⫻ 105 0 5.25 ⫻ 103 7.63 ⫻ 104 9.55 ⫻ 104 0 6.61 ⫻ 104 8.05 ⫻ 104 5.55 ⫻ 104
a Cells were grown in HJGS. Glycine was added to cultures at an OD660 of 0.5, and the cultures were incubated for an additional 2 h. b ⫺, 100 ⍀; 0, 200 ⍀; ⫹, 400 ⍀. c ⫺, 1.4 kV; 0, 1.65 kV; ⫹, 1.9 kV. d ⫺, 1 h; 0, 2 h; ⫹, 3 h. e ⫺, pH 4.0; 0, pH 4.5; ⫹, pH 5.0.
led to a further decrease in the EPB pH. Finally, a VC(MgCl2) of ⫺15.5% led to omission of magnesium from the EPB. The results of the final multifactorial experiment, in which four factors were tested, are shown in Table 3. n was 6.7 ⫻ 104 Tf/g and a was 5.5 ⫻ 104 Tf/g, which suggested that optimal conditions were again found within the test parameters. Interactions between certain factors were indicated by elevated VC values; e.g., VC(⍀, kV) was ⫺93%, and VC(pH, hours in glycine) was ⫺94%. Linear experiments were designed to optimize each pair of variables. Treatment for 1 h in the presence of 10% glycine and subsequent washing and resuspension of cells in pH 4.5 buffer resulted in the greatest number of transformants (data not shown). The results of experiments in which the relationship between the resistance setting and the voltage applied was examined were more difficult to interpret, as shown in Fig. 1. When the resistance was 400 ⍀, there was a clear peak in transformability at 1.25 kV, and the efficiency decreased at higher or lower voltages. The greatest number of transformants was obtained at 1.45 kV and 200 ⍀; however, the number of transformants obtained decreased sharply as the voltage was increased or decreased. The next highest number of transformants was obtained at a voltage of 1.60 kV, with high numbers on either side. Thus, additional experiments were performed with a resistance of 200 ⍀ and a voltage of
FIG. 1. Relationship between the number of transformants obtained per milligram of pNZ123 DNA and the voltage of the electroporator. Electroporation was performed with 200 ng of added DNA at the voltages indicated and at levels of resistance of 100 ⍀ (‚), 200 ⍀ (䊐), and 400 ⍀ (E). Tf, transformants.
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FIG. 2. Relationship between the amount of pNZ123 DNA in the electroporation mixture and the number of transformants obtained. Electroporation was performed as described in Materials and Methods. Tf, transformants.
1.60 kV. The viability of S. salivarius in response to electroporation was 50% when the starting concentration was 1010 cells/ ml, measured as described above. The relationship between the amount of plasmid DNA present in the electroporation cuvette and the number of transformants obtained is shown in Fig. 2. The highest number of transformants was obtained with 1 g of pNZ123. The optimal conditions for preparation of cells and electroporation were as follows. Cultures used to prepare competent cells were initiated by adding 1% overnight precultures. Glycine was then added to a final concentration of 10% when the culture OD660 was 0.5. The cells were incubated for 1 h at 37°C and collected by centrifugation. They were washed twice in ice-cold EPB (5 mM potassium phosphate [pH 4.5], 0.4 M sorbitol, 10% glycerol), concentrated 50-fold, frozen in an ethanol-dry ice bath, and then stored at ⫺80°C until they were used. Cells were thawed on ice, combined with 1 g of plasmid, and transferred to a prechilled 1-mm electroporation cuvette, and electroporation was performed with the standard settings (1.60 kV and 200 ⍀). After the electric pulse, the cells were diluted in 1 ml of ice-cold growth medium containing 0.4 M sorbitol and incubated for 3 h at 37°C. Aliquots were spread onto agar plates, and transformants were counted after 48 to 72 h of incubation. Using this procedure, we reproducibly obtained approximately 105 S. salivarius ATCC 25975 transformants/g of pNZ123 DNA. After close to 2 years of storage at ⫺80°C, S. salivarius cells retained their electroporation competence, and their transformation efficiency was not significantly different from the transformation efficiency of freshly
TABLE 4. Efficiency of transformation of pNZ123 into electroporation-competent S. salivarius ATCC 25975 and other strainsa S. salivarius strain
Transformation efficiency (transformants/g of pNZ123)
ATCC 25975 ................................................... ATCC 25975c .................................................. ATCC 7073 ..................................................... ATCC 13419 ................................................... 30.1................................................................... a
2.2 ⫻ 105 ⫾ 1.3 ⫻ 105b 8.4 ⫻ 104 ⫾ 3.7 ⫻ 104 9.3 ⫻ 105 ⫾ 5.0 ⫻ 105 5.3 ⫻ 104 ⫾ 1.6 ⫻ 104 5.3 ⫻ 102 ⫾ 3.8 ⫻ 101
All cells were transformed with 1 g of pNZ123 DNA. Average ⫾ standard deviation for three to six samples of each S. salivarius preparation. c This preparation had been stored at ⫺80°C for 20 months at the time of testing. b
FIG. 3. (A) Southern blot analysis of plasmid preparations from spectinomycin-resistant transformants probed with plasmid pDL278. Lane 1, pDL278: lane 2, S. salivarius ATCC 25975; lane 3, L. lactis LM 0230; lane 4, P. acidilactici SMQ-250; lane 5, S. sanguis ATCC 10556; lane 6, S. sobrinus 6715; lane 7, S. vestibularis ATCC 49124; lane 8, S. thermophilus ATCC 19258; lane 9, S. thermophilus DT1 (now known as S. thermophilus SMQ-310); lane 10, S. salivarius ATCC 25975 Spr Cmr. (B) Southern blot analysis of plasmid preparations from chloramphenicol-resistant transformants probed with plasmid pNZ123. Lane 1, pNZ123; lane 2, S. salivarius ATCC 25975; lane 3, L. lactis LM 0230; lane 4, S. vestibularis ATCC 49124; lane 5, S. mutans XS123; lane 6, S. thermophilus DT1 (now known as S. thermophilus SMQ-310); lane 7, S. salivarius ATCC 25975 Spr Cmr.
prepared cells (Table 4). We also tested the procedure with other strains of S. salivarius and found that all of the strains could be transformed, but the transformation efficiencies were lower with strain ATCC 13419 and fresh isolate 30.1 and greater with strain ATCC 7073 than with S. salivarius ATCC 25975 (Table 4). The transformation efficiencies could be improved by optimizing the electroporation conditions for individual strains. Plasmid stability and double transformation. The stabilities of plasmid markers in transformants were measured as described above. After growth in the absence of selective pressure, pDL278 and pNZ123 were maintained in all S. salivarius transformants tested after 20, 40, and 60 generations. It was possible to introduce both pDL278 and pNZ123, which produced a double transformant of S. salivarius (Fig. 3). The efficiency of pNZ123 introduction was the same whether the cells contained pDL278 or not. However, the efficiency was reduced drastically if electroporation was conducted with both plasmids at the same time or if pDL278 was introduced into cells harboring pNZ123 (1 to 2 transformants/g of DNA). Whether antibiotic selection was simultaneous or sequential made no difference to transformation efficiency. Transformation of other bacteria. The technique described above was applied to eight other bacterial species with pNZ123 and pDL278. The results of these experiments are shown in Table 5. Each strain tested was transformed with at least one of the plasmids. The presence of specific plasmids in the various transformants was confirmed by Southern hybridization in which pNZ123 or pDL278 was the probe, as described above (Fig. 3). The sizes of the recovered plasmids corresponded to the original sizes of the transforming plasmids, indicating that
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TABLE 5. Efficiencies of transformation of glycine-treated streptococci and related bacteria Transformation efficiency (transformants/g of DNA)a
Bacterium
pNZ123
S. salivarius ATCC 25975 S. sanguis ATCC 10556 S. sobrinus 6715 S. thermophilus SMQ-310 S. thermophilus ATCC 19258 S. vestibularis ATCC 49124 S. mutans XS123 L. lactis LM 0230 P. acidilactici SMQ-250 a
pDL278 5
1.7 ⫻ 10 0 0 8.8 ⫻ 103 19 1.7 ⫻ 105 0 3.7 ⫻ 104 0
2.8 ⫻ 103 1.0 ⫻ 103 39 2.6 ⫻ 103 23 8.4 ⫻ 104 4.6 ⫻ 103 9.3 ⫻ 103 112
All cells were transformed with 1 g of plasmid DNA.
the plasmids had not undergone any overt size modification following transformation into and replication in the new host. Although S. mutans XS123 was not transformed with pDL278 by electroporation techniques used for other strains of S. mutans (1), we were able to transform this strain with the technique described in this paper. A technique for introducing DNA into S. sobrinus via mobilization has been described (1a) because electroporation of this bacterium is very difficult (10). The transformation efficiency which we measured was very low; however, the method described in this paper can serve as a starting point to improve efficiency. This technique has also been used to introduce DNA into Lactobacillus species when all other avenues had been exhausted (9a), further supporting the general utility of the technique. In this study we developed a new method for preparing electroporation-competent cells and found that it may be applied to a variety of gram-positive bacteria. The technique was optimized for S. salivarius ATCC 25975, which had never been transformed previously despite numerous attempts with a wide variety of protocols. ACKNOWLEDGMENTS Medical Research Council of Canada grants MT6979 and MT11276 supported this study, and M.F. is a scholar of the Fonds de la Recherche en Sante´ du Que´bec. REFERENCES 1. Benchabane, H., and M. Frenette (Universite´ Laval). Personal communication. 1a.Buckley, N. D., L. N. Lee, and D. J. LeBlanc. 1995. Use of a novel mobilizable vector to inactivate the scrA gene of Streptococcus sobrinus by allelic replacement. J. Bacteriol. 177:5028–5034. 2. Caldwell, S. L., D. J. McMahon, C. J. Oberg, and J. R. Broadbent. 1996. Development and characterization of lactose-positive Pediococcus species for milk fermentation. Appl. Environ. Microbiol. 62:936–941. 3. David, S., G. Simons, and W. M. De Vos. 1989. Plasmid transformation by electroporation of Leuconostoc paramesenteroides and its use in molecular cloning. Appl. Environ. Microbiol. 55:1483–1489. 4. Dunny, G. M., L. N. Lee, and D. J. LeBlanc. 1991. Improved electroporation and cloning vector system for gram-positive bacteria. Appl. Environ. Microbiol. 57:1194–1201. 5. Hammes, W., K. H. Schleifer, and O. Kandler. 1973. Mode of action of glycine on the biosynthesis of peptidoglycan. J. Bacteriol. 116:1029–1053.
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