Electrotransformation of Lactobacillus acidophilus Group A1

ELSEVIER

FEMS Microbiology Letters 138 (1996) 233-237

Electrotransformation

of Lmtobacillus acidophilus Group Al

D. Carey Walker a, Kenji Aoyama b, Todd R. Klaenhammer a Department h Department

of Microbiology, qf Food Science,

a3b3 *

Southeast Daiy

Foods Research Center, North Carolinn

State Unirer.sity,

Raleigh,

NC 27695-7624,

USA

Southeast Dairy

Foods Research Center, North Carolinn

State University,

Raleigh,

NC 27695-7624.

USA

Received 2 January 1996; revised 18 March 1996; accepted 2 I March 1996

Abstract Two strains of Lmrobacillus acidophilus Group Al, the neotype ATCC 4356 and a human isolate NCFM-N2, widely used as a dietary adjunct in milk and cultured dairy products, were transformed with plasmid DNA by electroporation. The transformation characteristics exhibited by the two L. acidophilus strains were found to differ markedly even though they appeared similar at the genomic level based on the DNA patterns of SmuI restriction fragments. To our knowledge, this is the first report of a consistent, reproducible transformation system of Lactobacillus acidophilus strains comprising the Al DNA homology group. Keytvords:

Transformation; Electropordtion;

Lcrctobncillus

acidophilus;

1. Introduction As normal inhabitants of the mammalian gastrointestinal tract, Lmrobacillus species have been implicated in health-related effects including immunostimulation, improved lactose digestion, cholesterol reduction, and a reduction in fecal tumorigenic metabolic activity [ 11. Informed decisions concerning the veracity of health-related claims, and a fundamental understanding of the molecular basis of these and other potentially beneficial activities, will depend upon the development of appropriate genetic techniques for working with Luctobacillus. A description of the phenotypically heterologous L. acidophilus species was provided in 1970 with the designation of a neotype strain ATCC 4356 [2]. More recently the L. acidophilus species was subdi-

* Corresponding author.

Plasmid

vided into six DNA homology groups [3] with Johnson’s group Al retaining the designation L. acidophilus [4], and groups Bl and B2 assigned as L. gasseri [5] and L. johnsonii [6], respectively. The classification of Lactobacillus strains is now augmented by the techniques of SDS-PAGE protein pattern analysis, and species-specific rRNA-targeted oligonucleotide probes [7], which have confirmed a close relationship between the intestinal species of lactobacilli. Although numerous lactobacilli have been successfully transformed by electroporation, we have been unable to transform the neotype strain of the L. acidophilus Al group, ATCC 4356, using published protocols [8,9]. In order to achieve effective and reproducible electrotransformation of the A 1 group we modified a protocol that has proven successful with L. helceticus CNRZ 32 [lo]. The new protocol was used to successfully electroporate pGK12 [ 111 into both the neotype L. acidophilus ATCC 4356

0378-1097/96/$12.00 0 1996 Federation of European Microbiological Societies. All rights reserved PII SO378-1097(96JOOl 15-2

and L. ucidophilus NCFM-N2 which is used extensively as a dietary adjunct in milk. NCFM-N2 hybridizes to the type Al-oligonucleotide probe (Lba) described by Pot et al. [7]. thus confirming its classification within the A I group [ 121. The transformation effectiveness of the new L. acidophilus electroporation protocol was also tested against the neotype strains of both L. johnsonii and L. gusseri and compared with transformation frequencies obtained from a general Lactohacillus transformation protocol.

2. Materials and methods

Louis. MO, pH 7.0 using 2 N NaOH). The cell pellet was resuspended in 1.6 ml in cold electroporation buffer, divided into 800 ~1 aliquots, and kept on ice. One aliquot was mixed with 10 pg of plasmid DNA and transferred to a chilled cuvette (0.4 cm electrode gap). After 5 min on ice, the suspension was electroporated at 2.45 kV, 2000 and 2.5 p,f (GenePulserTM, Biorad Laboratories. Richmond, CA). The cuvette was placed back on ice for 5 min, transferred to 8.0 ml of recovery medium (MRS broth containing 10.0 mM CaCl, and 0.5 M sucrose), and incubated at 37°C for 3 h prior to plating on the selective medium (MRS agar containing 10.0 mM CaCl? and 5.0 or 7.5 pg/ml chloramphenicol).

2. I. Bacterial strains

2.3. S-luver- treutment

The neotype strains L. acidophilus ATCC 4356. L. gasseri ATCC 33323, and L. johnsanii ATCC 33200 were obtained from the American Type Culture Collection, Rockville, MD. L. acidophilus NCFM-N2 and the closely related strains RL8K and Isol. 6 were obtained from our NCK culture collection. All bacterial cultures were stored at - 70°C in Lactobacilli MRS broth (Difco Laboratories, Inc., Detroit, MI) containing 20% glycerol.

A 100 ml MRS broth culture was incubated at 37°C to an OD,(,,, of 0.65. The cells were centrifuged, resuspended in 5.0 ml of 5.0 M LiCl,, and held on ice for I h. The cells were rinsed 3 X in 3.5 X SMEB and electroporated following the method of Luchansky et al. [8].

2.2. Electrotransformation Prior to each transformation experiment the Lactobacillus strains were transferred from - 70°C stock cultures to MRS broth (Difco Laboratories, Detroit. MI) and incubated overnight at 37°C. In one case. the method of Luchansky et al. [8], with 3.5 X SMEB buffer was used for electroporation. Fresh MRS broth cultures were inoculated (1 .O%b) and harvested at OD,, of 0.65. Electrocompetent cells from the new L. ucidophilus Al protocol. were prepared from fresh MRS cultures propagated for 1.5 h in broth containing 1.0% glycine or 20 mM DL-threonine. A 1.0% inoculum was then made to 200 ml of MRS broth containing I .O% glycine or 20 mM DL-threonine and the cells propagated to an ODs,,, of 0.25-0.30. The cells were placed on ice (5 min) and centrifuged. The pellet was held on ice and rinsed 4 X with 200 ml of ice-cold electroporation buffer (0.1 mM HEPES (N-[2-hydroxyethyl]piperazine-N’-[2-ethanesulfonic acid]), 0.5 M sucrose (Sigma Chemical Company, St.

2.4. DNA manipulations Plasmid DNA used in the electroporation experiments was isolated from 40 ml Escherichia coli cultures using the Qiagen Plasmid Midi Kit as outlined by the manufacturer (Qiagen Inc, Chatsworth. CA). Plasmid isolation and analysis of Lactobacillus transformants and the comparison of L. acidophilus strains by pulsed-field gel electrophoresis were conducted as described previously [ 131.

3. Results and discussion Transformation experiments with the L. ucidophilus strains using the protocol of Luchansky et al. [8] averaged 1.2 X 10” NCFM-N2 transformants/pg plasmid DNA (Table 1). No ATCC 4356 transformants were recovered using the method. In an attempt to break the transformation barrier of ATCC 4356, we evaluated various modifications of the Bhowmik and Steele [IO] electroporation protocol. First the concentration of pGK12 was increased from 1.0 pg to 10.0 pg per 800 ~1 cell suspension.

D.C. Walker et al./

FEMS Microbiolog!

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235

Table 1 Lmtobacillus

transformation

LactobaciNus

strain

frequencies/pg Luchansky Cm 5.0 *

L. acidophiius L. acidophilus L. johnsonii

a The b The ‘ The d The ’ The ’ The ’ The

pGKl2

DNA in response to variations

et al. ’ Cm 7.5 ’

Al + glycine Cm 5.0

in the electroporation b

Cm 7.5

protocol

Al + DL-threonine ’ Cm 5.0

Cm 7.5

NCFM-N2

ATCC 4356 ’ ATCC 33200 s

basic transformation protocol of Luchansky et al. [8] using a 3.5 X sucrose:MgClz electroporation buffer (SMEB) L. acidophilus Al electroporation protocol using 1.0% glycins as the cell wall destabilizer. L. ucidophilus Al electroporation protocol using 20 mM Dt_-threonine substituted for 1.0% glycine. selection medium was MRS agar with 10 mM CaClz and 5.0 pg chloramphenicol/ml. selection medium was MRS agar with 10 mM CaCl, and 7.5 @g chloramphenicol/ml. transformation frequencies for this strain are an average of at least three replications. transformation frequencies for this strain are an average of at least two replications.

since higher DNA concentrations increase transformants in a linear fashion without reaching saturation levels [14]. Second, 0.5 M sucrose was added to the recovery medium to promote the survival of cells osmotically destabilized by glycine, as per Ho10 and Nes [ 151. With these modifications, pGK12 transformants were recovered in both ATCC 4356 and NCFM-N2 (Table 1). Plasmid pGK 12 was recovered from the transformants of both strains (data not shown).

Various factors were manipulated in an effort to increase transformation frequencies. It was possible that the S-layer proteins of the Al homology group, found in NCFM-N2 and ATCC 4356 [16], presented a formidable barrier to plasmid entry. S-layer proteins are removed from Lactobacillus strains using lithium chloride [ 171 with some loss in cell viability. Attempts to electrotransform L. acidophilus NCFMN2 or ATCC 4356 with pGKl2, following treatment with 5.0 M LiCl, were unsuccessful. No alternative

Mol. Weight ATCC 4356 NCFM-lsol. 6 NCFM-N2

Fig. 1. Pulsed-field gel electrophoresis (PFGEl of total DNA isolated from Lacrobacillus acidophilus strains. Total DNA was isolated in situ, digested with SmaI, and separated by pulse field gel electrophoresis. The PFGE conditions were 200 volts, l-20 s switching times, and 22 h total run time. RL8K was first isolated in 1975 and is identical in its SmaI restriction pattern to NCFM which is the progenitor of two isolates obtained from commercial sources, NCFWN2 and NCFM-Isol. 6.

strategies to remove the S-layer proteins prior to electroporation were attempted. The effects of various cell wall weakening agents is variable among different Lactohacillus strains. Therefore, 20 mM DL-threonine was evaluated as an alternative agent to glycine for cell wall destabilization prior to electroporation (Table 1). The incorporation of 20 mM DL-threonine in the MRS growth medium resulted in a transformation frequency of I .6 X lO’/pg pGKl2 in NCFM-N2, similar to the frequencies obtained using 1.0% glycine. Use of DL-threonine with ATCC 4356 reduced the number of transformants at least ten-fold. Therefore, while either glycine or DL-threonine can be used effectively with NCFM-N2. glycine should be used for ATCC 4356. The L. crcidopkilus strains were also examined for restriction barriers. Restriction modification (R/M) systems are widely distributed in lactic acid bacteria and can be circumvented by heat shock [ 181 or prior passage of the transforming replicon through an R+/M+ host [19]. Following a 50°C heat shock, the transformation rate for both NCFM-N2 and ATCC 4356 dropped significantly relative to the nonshocked controls (data not shown). In addition. when pGK12 was passed through NCFM-N2 and ATCC 4356 and used in second round electroporation experiments. no increase in the frequency of transformation was observed. Therefore. an R/M system was not implicated in the L. ucidopkilus Al strains by these preliminary experiments. Lastly. a decrease in chloramphenicol from 7.5 Fg/ml to 5.0 pg/ml resulted in a slight increase in the transformants recovered. The effect was most pronounced with ATCC 4356 (Table 1) and underscored the difference in the electroporation competencies of the two L. acidopkilus strains. In this study, L. acidopkilus ATCC 4356 and NCFM-N2 exhibited marked differences in their ability to accept DNA by electroporation. However, PFGE comparisons of SmaI restriction fragments for the two strains indicated a marked similarity in genomic organization (Fig. l), as only one band appeared different in size. In an effort to examine if the Smul band difference was a characteristic that might fluctuate over time, leading to variable transformation frequencies, we evaluated the clonal isolates of NCFM-N2 back 20 years. No variation was

identified and the genetic lineage of the RL8K. NCFM, and NCFM-N2 strains was confirmed. Three other L. ucidopkilus A I -type strains showed patterns identical to either ATCC 4356 or NCFM-N2 (data not shown). The results suggested a conserved genomic organization between the L. acidopkilus strains used in this study and those found in industrial products. Whether or not these are identical strains is unknown. Nevertheless. these strains varied markedly in their transformation characteristics supporting strain-by-strain optimization to achieve maximum efficiencies. The new L. ucidopkilus Al protocol was also evaluated with the neotype strains of L. gusset-i and L. joknsonii and compared to previous methods (Table 1). Based on these results, the basic electroporation protocol of Luchansky et al. [8] is recommended for L. gnsseri and L. joknsonii. During the course of this study the frequency of NCFM-N2 transformation generated with Luchansky’s protocol varied between 10’ and 10’ transformants/pg pGK12 (data not shown). The new Al protocol, with either DLthreonine or glycine, as the cell wall destabilizer. gave more consistent results at levels of 10’ transformants/pg. L. acidopkilus ATCC 4356 transformants were only recovered using the Al protocol and were consistently generated with glycine. These results indicate that plasmid DNA can be efficiently introduced into L. ucidopkilus (Al homology group) by electroporation when steps are taken to reduce the integrity of the cell wall and protect the osmotically fragile cells. The ability to consistently transform this important Luctohacillus species breaks an important barrier for future genetic investigations of health-related targets such as adherence mechanisms, pathogen exclusion, and immunocompetence.

Acknowledgements Paper number FSR95-55 in the Journal the Department of Food Science, North State University, Raleigh, NC 27695-7624. This study was supported in part by Carolina Dairy Foundation. K. Aoyama ported as a visiting scientist at NCSU Brand Milk Products Co., Ltd. Japan.

Series of Carolina the North was supby Snow

D.C. Wulker et rd./ FEMS Microhiologx

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