Optimization of technical conditions for the transformation of Lactobacillus acidophilus strains by electroporation

AIMS: To optimize the conditions for electroporating foreign plasmid DNA into Lactobacillus acidophilus ATCC 43121. METHODS AND RESULTS: The conditions of electroporation were optimized to improve the transformation efficiency. Plasmid pNZ123 containing multicloning site and chloramphenicol resistance was employed to construct a cloning vector. The optimum electroporation conditions for the maximum transformation efficiency were a pulse strength of 12.5 kV cm(-1), a pulse number of 10, a pulse interval of 500 ms, and pNZ123 plasmid DNA concentration of 25 ng microl(-1). Under the optimum conditions the transformation efficiency of L. acidophilus ATCC 43121 was 1.84 +/- 0.13 x 10(4) (+/- standard error of measurements) CFU per mug of plasmid DNA. Other strains of L. acidophilus showed transformation efficiencies ranging from 1.38 +/- 0.02 x 10(4) to 9.32 +/- 0.54 x 10(4) under these conditions. A green fluorescent protein (GFP) was successfully expressed and detected by fluorescence microscopy when the pKU::slpA-GFP, pNZ123 containing GFP gene, was transformed in L. acidophilus ATCC 43121 under the optimum conditions. CONCLUSIONS: The results suggest that electrical parameters, antibiotic concentration, and host specificity play important roles to determine transformation efficiency of lactobacilli. The optimum conditions for the transformation of L. acidophilus ATCC 43121 may be applied to improve transformation efficiency of other lactobacilli. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimized conditions for electrotransformation may provide a mean to improve the introduction of foreign DNA into L. acidophilus to be used as a vehicle for a heterologous protein expression.     

Transformation of Bacillus thuringiensis by electroporation

Plasmids were transformed by electroporation into various strains of Bacillus thuringiensis with frequencies of up to 10(5) transformants/micrograms. pC 194 transformed all strains tested at a high frequency and cells could be stably transformed with pC194 and pUB110 simultaneously by electroporation with a frequency of 10(2) pC194+ pUB110 transformants/micrograms DNA. Low transformation frequencies observed with some plasmids, especially those grown initially in Escherichia coli, could be increased by passage through B. thuringiensis, B. thuringiensis var. israelensis and in acrystalliferous mutant of the same strain transformed at frequencies of 10(4)-10(5)/micrograms DNA with most of the plasmids tested. A cloned israelensis 27-kDa delta-endotoxin gene was introduced into the israelensis acrystalliferous mutant and a kurstaki acrystalliferous mutant by electroporation. Both transformants were shown to express the endotoxin gene and to be toxic to Aedes aegypti larvae.     

Molecular cloning of, and phylogenetic analysis of, an actin in Naegleria fowleri

Abstract Two strains of Lactobacillus acidophilus Group A1, 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 Sma I restriction fragments. To our knowledge, this is the first report of a consistent, reproducible transformation system of Lactobacillus acidophilus strains comprising the A1 DNA homology group.     

Cloning, phenotypic expression, and DNA sequence of the gene for lactacin F, an antimicrobial peptide produced by Lactobacillus spp

Lactacin F is a heat-stable bacteriocin produced by Lactobacillus acidophilus 11088. A 63-mer oligonucleotide probe deduced from the N-terminal lactacin F amino acid sequence was used to clone the putative laf structural gene from plasmid DNA of a lactacin F-producing transconjugant, L. acidophilus T143. One clone, NCK360, harbored a recombinant plasmid, pTRK160, which contained a 2.2-kb EcoRI fragment of the size expected from hybridization experiments. An Escherichia coli-L. acidophilus shuttle vector was constructed, and a subclone (pTRK162) containing the 2.2-kb EcoRI fragment was introduced by electroporation into two lactacin F-negative strains, L. acidophilus 89 and 88-C. Lactobacillus transformants containing pTRK162 expressed lactacin F activity and immunity. Bacteriocin produced by the transformants exhibited an inhibitory spectrum and heat stability identical to those of the wild-type bacteriocin. An 873-bp region of the 2.2-kb fragment was sequenced by using a 20-mer degenerate lactacin F-specific primer to initiate sequencing from within the lactacin F structural gene. Analysis of the resulting sequence identified an open reading frame which could encode a protein of 75 amino acids. The 25 N-terminal amino acids for lactacin F were identified within the open reading frame along with an N-terminal extension, possibly a signal sequence. The lactacin F N-terminal sequence, through the remainder of the open reading frame (57 amino acids; 6.3 kDa), correlated extremely well with composition analyses of purified lactacin F which also predicted a size of 51 to 56 amino acid residues. Molecular characterization of lactacin F identified a small hydrophobic peptide that may be representative of a common bacteriocin class in lactic acid bacteria.     

Conjugative transfer of a shuttle plasmid for Escherichia coli to Mycobacterium smegmatis

Plasmid DNAs were introduced by electroporation into Bacillus subtilis PB1424 as an alternative to competent-cell or protoplast transformation. The maximum electroporation efficiency was 10(4) transformants/microgram DNA. Parameters including growth phase of cells, ionic strength of the suspending medium, concentration and size of plasmid DNAs, amplitude and duration of the pulse, were evaluated in order to determine conditions that improved transformation efficiency.     

球形芽孢杆菌TS—1原生质体电诱导质粒转化研究

This report gave the best conditions of Bacillus sphaericus Ts-1 protoplast-plasmid pHV33 electroporation. The highest transformation frequency and transformation efficiency induced by three pulse of 21 KV/cm and 10 microseconds duration applied at an interval of one sec., was 2.44 x 10(2) transformants/micrograms DNA and 3.16 x 10(-6) respectively. The saturated concentration of DNA absorbed by the protoplast was 5 micrograms DNA/10(9) cells/ml. By means of this method, pJB417, a recombinant mosquito larvicide clone, was introduced into B. subtilis 168M and B. sphaericus Ts-1. The transformants of B. subtilis 168M with biocide activity were obtained, but the toxicity of B. sphaericus Ts-1 was not increased.     

High efficiency electroporation of intact Corynebacterium glutamicum cells

High-frequency electroporation of whole Corynebacterium glutamicum cells without enzymatic pretreatment was achieved. Under optimized conditions concerning growth stage, washing of cells, cell concentration and pulse parameter transformation efficiencies of far more than 10(7) transformants per microgram pWST4B plasmid DNA were reached. Using electroporation, linearised and subsequently religated plasmid as well as chimeric ligase reaction products were directly introduced into C. glutamicum with reasonable efficiencies. Electrotransformation efficiency was reduced about 10(5)-fold for plasmid DNA cycled through E. coli JM83. Restriction deficient mutants of C. glutamicum were isolated which could be efficiently transformed with foreign DNA.     

Electrotransformation of Lactobacillus delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis with various plasmids.

We describe, for the first time, a detailed electroporation procedure for Lactobacillus delbrueckii. Three L. delbrueckii strains were successfully transformed. Under optimal conditions, the transformation efficiency was 10(4) transformants per microg of DNA. Using this procedure, we identified several plasmids able to replicate in L. delbrueckii and integrated an integrative vector based on phage integrative elements into the L. delbrueckii subsp. bulgaricus chromosome. These vectors provide a good basis for developing molecular tools for L. delbrueckii and open the field of genetic studies in L. delbrueckii.     

Plasmid transformation of Bacteroides spp. by electroporation

Transformation of Bacteroides spp. with a variety of plasmid DNAs was accomplished using electroporation. The standard transformation assay system used to deduce the optimal electroporation parameters employed a 50-to 100-fold concentrated cell suspension of mid-logarithmic phase Bacteroides fragilis strain 638 and the 5.4-kb clindamycin resistance (Ccr) vector, pBI191. A variety of electroporation buffers were used successfully in transformation experiments but of these, 1 mM MgCl2 in 10% glycerol was superior. The incorporation of MgCl2 was essential for optimum viability prior to electroporation and for optimum transformation. Transformants were routinely obtained using 5-ms pulses over a range of field strengths from 5 to 12.5 kV/cm, with a maximum of greater than 10(6) micrograms-1 DNA at 12.5 kV/cm. The number of transformants increased linearly with respect to DNA concentration over the range 0.01-2 micrograms tested. Recovery of transformants required an expression period of up to 2.5 h following exposure to the electric field. This period, however, was dependent on the antibiotic resistance marker used for selection of transformants, with a significantly shorter incubation required when chloramphenicol rather than clindamycin was used in the selective medium. The effect of the DNA source on transformation was tested using the shuttle vector pFD288. Plasmid DNA isolated from Bacteroides uniformis, Bacteroides ovatus, or Bacteroides thetaiotaomicron transformed B. fragilis 638 at frequencies 7.5- to 12.5-fold less than those observed for controls with homologous DNA. Further reductions were seen with Escherichia coli purified pFD288, which transformed at 1000-fold lower frequencies. Finally, using homologous pFD288 or pBI191 isolated from strain 638, several strains of B. fragilis, B. uniformis, and B. ovatus were transformed successfully without modification of the standard assay system. Two strains each of B. thetaiotaomicron and Bacteroides ruminicola were not transformed using the methods described here.     

Improvement of transformation and electroduction in avermectin high-producer,<Emphasis Type="Italic">Streptomyces avermitilis</Emphasis>

Factors affecting the PEG-mediated transformation and electrotransformation of Streptomyces avermitilis protoplasts, an industrial avermectin high-producer, were evaluated. The maximum protoplast transformation efficiency under optimum conditions with PEG was 3 x 106 transformants per microg plasmid pIJ702 DNA. The efficiency of electrotransformation with the same plasmid the intact cells grown in medium with 0.5 mmol/L CaCl2, suspended in buffer with 0.5 mol/L sucrose +1 mmol/L MgCl2, and pulsed at an electric field strength of 10 kV/cm, 800 ohms, 25 microF, was of 2 x 10(3) transformants per microg DNA. When the cells were electroporated after mild lysozyme-treatment, the efficiency was up to 10(4) transformants per microg DNA. Electroporation of protoplasts and germlings had a lower efficiency (10(2) transformants per microg DNA). We report that electroporation under optimum conditions can be used for direct transfer of nonconjugative plasmid pIJ699 between two different Streptomyces species, S. avermitilis and S. lividans.     

Electroporation: application to human lymphoid cell lines for stable introduction of a transactivator gene of human T-cell leukemia virus type I.

Conditions were developed for stable introduction of foreign DNA into human lymphoid cell lines by electroporation. To introduce stably the p40 gene of human T-cell leukemia virus type I (HTLV-I) into the human lymphoid cell line Jurkat, the p40 expressing plasmid, pMAXRHneo-1, which carries the neo resistant gene, was transfected into Jurkat cells at a voltage of 2500 V and capacitance of 21.7 microF, and stable transformants were screened for neo (G418) resistance. The frequency of transformants was more than one per 2 x 10(5) cells used initially. Clones that were resistant to G418 were shown to have the p40 gene integrated into the host genome and to express mRNA and protein from the introduced plasmid. Expression of p40 in the transformed Jurkat cells was also confirmed by testing the trans-activating effect of HTLV-I enhancer by p40. High frequencies of stable transformations of 10(-4) to 10(-6) were also reproducibly obtained by electroporation of the human T cell lines HSB-2 and TALL-1, a human B cell line Raji, a human monocytic cell line U937, and a human erythroleukemia cell line K562. These results demonstrate that electroporation is a very efficient method for introducing foreign DNA into human lymphoid cell lines.     

A highly efficient electroporation system for transformation of Yersinia

The various pathogenic Yersinia species are not readily and efficiently transformed by classical methods. For this reason, the electroporation technique was applied for genetic transformation of these species. Using optimal conditions, we were able to transform the six Yersinia strains studied with the two most widely used groups of plasmids: pSU2718 (a pACYC184 derivative) and pK19 (a pUC19 derivative). Only Yersinia enterocolitica (Y. e.) serotype 0:8 gave poor results (less than 5 x 10(2) transformants/microgram) DNA). Electrical transformation of the other species resulted in high efficiencies, up to 10(5) transformants/microgram DNA for Y. e. serotypes 0:3 and 0:9, 10(6) for Y. pseudotuberculosis and 10(7) for Y. pestis. The results varied for each strain with the type of plasmid used. Neither the introduced foreign plasmid nor the resident 72-kb virulence plasmid underwent detectable deletions. Transformation was most efficient with supercoiled DNA, decreasing by one and four orders of magnitude for relaxed circular and linearized plasmids, respectively. The ability to easily and efficiently transfer plasmid DNA via electroporation will greatly facilitate the application of recombinant DNA technology for direct cloning and analysis of significant genes into Yersinia.     

Improved method for electroporation of Staphylococcus aureus

We have developed a significantly improved method for the electroporation of plasmid DNA into Staphylococcus aureus. The highest transformation efficiency achieved with this procedure was 4.0 x 10(8) transformants per microgram of plasmid pSK265 DNA. This represents a 530-fold improvement over the previously reported optimum efficiency of 7.5 x 10(5) transformants per microgram of plasmid DNA after electroporation of S. aureus cells [9]. Identical results were obtained when electrocompetent cells, which had been stored frozen at -80 degrees C, were used. The improved efficiency is due primarily to the use of a modified medium (designated as B2 medium) and secondarily to the use of 0.1-cm cuvettes. Several other plasmids (pI258, pMH109, and pSK270) were also electrotransformed into competent cells using our procedure, and for each plasmid, the transformation efficiency was significantly reduced compared to that observed when pSK265 DNA was used. With respect to plasmid pI258, the transformation efficiency was 3500-fold higher than that reported previously for transformation of this plasmid into S. aureus RN4220 [9]. The optimized electroporation procedure was less successful in transforming other staphylococci. Electrocompetent cells of S. aureus ATCC 29213 and S. epidermidis ATCC 12228 produced 5.5 x 10(5) and 5 x 10(3) transformants per microgram of pSK265 DNA, respectively.     

Genetic transformation of rifampicin resistance in Lactobacillus acidophilus

Lactobacillus acidophilus strain 100-33, originally isolated from swine faeces, was transformed to rifampicin resistance with DNA from spontaneous rifampicin-resistant mutants derived from it. Cells of the recipient strain were treated with lysozyme and mutanolysin, mixed with donor DNA and polyethylene glycol and grown on a regeneration medium overnight. After 48 h incubation, the numbers of rifampicin-resistant cells in the populations of regenerated cells were estimated from numbers of colonies. Efficiency of the lysozyme/mutanolysin treatment (the ratio of the number of osmotically fragile cells after the enzyme treatment to the initial cell number) was about 99%. The regeneration frequency of the enzyme-treated cells varied from 5 to 67%. The transformation frequency varied from about 0.2 X 10(-8) to 8.0 X 10(-8) transformants per regenerated cell per microgram DNA. To our knowledge, this method for genetic transformation is the first to be reported for a Lactobacillus strain.     

Transformation of Acidiphilium by electroporation and conjugation.

Two techniques, electroporation and conjugation, have been used to introduce the RK2-based broad-host-range plasmids pRK415 and pLAFR3 into strains of the bacterial genus Acidiphilium. Using electroporation, cells were also transformed with a series of chimeric plasmids constructed by cloning cryptic Acidiphilium plasmids into the Escherichia coli vector pBR328. Various parameters affecting electroporation were investigated. Transformation efficiency varied widely with different recipient strains. Growth at an elevated temperature (37 degrees C) prior to electroporation increased transformation efficiency 10-fold compared with growth at 32 degrees C. For three strains tested, optimum transformation efficiency was obtained with field strengths of 10-15 kV/cm. Transformation efficiency increased linearly with increasing DNA concentration up to 10 micrograms/mL. Transformation efficiencies in these experiments ranged up to 10(4) transformants/micrograms DNA. Mobilization of pRK415 and pLAFR3 from E. coli strain S17.1 into several Acidiphilium strains was achieved following incubation for 3 h on nutrient agar medium (pH 7.0). Conjugation frequencies in the range of 10(-5)-10(-9) per recipient cell were obtained. Conjugation frequency was also dependent on recipient strain.     

Improved transformation of Pseudomonas putida KT2440 by electroporation

Summary An electric field-mediated transformation (i.e. electroporation) was performed to determine optimal conditions for P. putida transformation. The effects of culture age, electroporation buffer composition, electric field strength, pulse time constant and DNA concentration on transformation efficiency were examined. When plasmid DNA of 8 to 11 kb in size was used with an electroporation buffer containing 1 mM HEPES (pH 7.0), maximum transformation efficiency of 1.0 × 10⁷ transformants/g DNA was obtained at field strength of 12 kV/cm with pulse time of 2.5 millisecond. A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude. A linear relationship was observed between growth phase and transformation efficiency up to OD₆₀₀ = 2.0. This reliable and simple method should be useful for introduction of plasmid DNA into intact P. putida cells.     

Electroporation of rapeseed protoplasts – transient and stable transformation

Protoplasts of Brassica napus hypocotyls were transfected using electroporation. Parameters such as discharge potential, protoplast density and buffer constituents were tested to determine the most suitable conditions for gene transfer. To monitor the introduction of DNA into protoplasts a plasmid containing the β-glucuronidase (EC 3.2.1.31), and the neomycin phospotransferase (EC 2.7.1.95) genes was used. By using this construct, expression of a screenable marker gene for transient expression analysis as well as an antibiotic resistance marker gene for selection of stable transformants were obtained. Refined electroporation conditions resulted in a frequency of 0.1% transiently transformed protoplasts. Microcalluses were cultured under selective conditions in a bead-type culture system. Resistant callus, with an absolute transformation frequency of 4.9 × 10⁻⁵ and a relative transformation frequency of 0.3% could be achieved. X-ray irradiation of newly electroporated protoplasts did not enhance absolute transformation frequencies. From some of the resistant calluses, transgenic plants could be regenerated which were characterized by molecular analysis.     

Transformation of alternan-producing strains of Leuconostoc by electroporation

Alternan-producing Leuconostoc mesenteroides strain NRRL B-1355 and its glucansucrase-negative derivative NRRL B-21414 were transformed by electroporation using four Gram positive-Gram negative shuttle vectors. Optimal conditions were 400 Omega and 10 kV cm(-1), resulting in transformation efficiencies of up to 3.5 x 10(4) per microg DNA. Relatively low copy numbers and native plasmids made it difficult to visualize the introduced plasmids on ethidium bromide-stained gels and, in some cases, on blot hybridizations. However, PCR analysis indicated that 95% of putative transformants carried plasmid sequences. Direct colony PCR was shown to work well for this system and also for transformants of L. mesenteroides subsp. cremoris.     

High efficiency electroporation of Pseudomonas aeruginosa using frozen cell suspensions

High efficiency transformation of Pseudomonas aeruginosa was achieved using frozen cell suspensions and high voltage electroporation. We have obtained frequencies as high as 5.8 x 10(8) transformants/micrograms of plasmid DNA using PA01 strain OT684 and a buffer of 15% glycerol-1 mM MOPS. The method allows for easy and reproducible production of frozen cell suspensions for rapid transformation of P. aeruginosa.