An efficient transformation method for <Emphasis Type="Italic">Bacillus subtilis</Emphasis> DB104

Bacillus subtilis strains are used for extracellular expression of enzymes (i.e., proteases, lipases, and cellulases) which are often engineered by directed evolution for industrial applications. B. subtilis DB104 represents an attractive directed evolution host since it has a low proteolytic activity and efficient secretion. B. subtilis DB104 is hampered like many other Bacillus strains by insufficient transformation efficiencies (≤10³ transformants/μg DNA). After investigating five physical and chemical transformation protocols, a novel natural competent transformation protocol was established for B. subtilis DB104 by optimizing growth conditions and histidine concentration during competence development, implementing an additional incubation step in the competence development phase and a recovery step during the transformation procedure. In addition, the influence of the amount and size of the transformed plasmid DNA on transformation efficiency was investigated. The natural competence protocol is “easy” in handling and allows for the first time to generate large libraries (1.5 × 10⁵ transformants/μg plasmid DNA) in B. subtilis DB104 without requiring microgram amounts of DNA.     

DNA-mediated transformation system in a bipolar basidiomycete, <Emphasis Type="Italic">Pholiota microspora</Emphasis> (<Emphasis Type="Italic">P. nameko</Emphasis>)

We cloned a gene encoding the succinate dehydrogenase iron-sulfur protein subunit (sip) from a bipolar mushroom, Pholiota microspora, and introduced a point mutation that confers carboxin resistance into this gene. Using this homologous selective marker and also a heterologous drug selective marker, the hygromycin B phosphotransferase gene (hph), we successfully constructed a DNA-mediated transformation system in P. microspora. Both these selection markers have high transformation efficiency: the efficiency of carboxin resistance transformation was about 88.8 transformants/μg pMBsip2 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 1.0 μg/ml carboxin, and the efficiency of hygromycin B resistance transformation was about 122.4 transformants/μg pMBhph1 DNA using 5 × 10⁶ protoplasts in regeneration plates containing 150 μg/ml hygromycin B. Southern hybridization analysis showed that the introduced sequence (mutant sip or hph) was integrated into the chromosomal DNA in these transformants with a copy number of one or more.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Electroporation of, plasmid isolation from and plasmid conservation in Clostridium acetobutylicum DSM 792

Procedures have been developed allowing recombinant DNA work with Clostridium acetobutylicum DSM 792. Electroporation was used to introduce plasmid DNA into exponentially growing clostridial cells and 6 × 10² transformants/μg DNA could be obtained at a time constant of 5.5 ms, 1.8 kV, 50 μF, and 600 Ω. The method also allowed the taxonomic group IV strain NI-4082 to be transformed (10¹ transformants/μg DNA). Plasmid preparation from recombinant clostridia was optimal when a modification of the alkaline lysis method was employed. It was also important to use cells from the mid-logarithmic growth phase. Recombinant strains could be easily preserved as spore suspensions; under all conditions tested plasmids were maintained. Received: 17 March 1998 / Received revision: 17 August 1998 / Accepted: 26 August 1998     

Genetic transformation of Clostridium botulinum hall a by electroporation

Summary A method was developed for the introduction of plasmids into Clostridium botulinum by electroporation. A 4.4 kb plasmid vector, pGK12, which contains genes for resistance to erythromycin (Em^r) and chloramphenicol (Cm^r) was electroporated into C. botulinum type A (Hall A). The highest transformation efficiency was obtained using midlog phase cells, 10% PEG 8000 as the electroporation solution, and 2.5 kV field strength. The transformation efficiency was highest (10³ transformants/g of DNA) when 1 g of plasmid DNA and 4 × 10⁸ CFU/ml of recipient cells were used. Plasmid DNA recovered from the transformants was indistinguishable from that introduced on the basis of restriction enzyme digestion and agarose gel electrophoresis.     

Transformation of spheroplasts and protoplasts of Corynebacterium glutamicum

Summary Spheroplasts were prepared from Corynebacterium glutamicum ATCC13032 by growing cells in the presence of glycine followed by digestion with lysozyme. Using pUL330 a spheroplast transformation system was established routinely yielding 10³ to 10⁴ transformants per g of plasmid DNA. Spheroplasts were converted into protoplasts after incubation with the lytic enzyme achromopeptidase in the presence of additional lysozyme. Protoplasts prepared by this method regenerated at efficiencies of 10 to 30%. A protoplast transformation system was established routinely yielding 10⁵ to 10⁶ transformants per g of plasmid DNA. The Escherichia coli Brevibacterium lactofermentum shuttle vector pUL62 prepared from E. coli could be introduced into spheroplasts of C. glutamicum after heat treatment at 48 to 49°C for 10 min.     

Efficient transformation ofKlebsiella oxytoca by electroporation

A protocol for the transformation ofKlebsiella oxytoca by electroporation was developed. Preparation of competent cells at early exponential phase was most critical to obtain a high transformation efficiency. The highest efficiency of 1.6 × 10⁶ transformants per μg DNA (pBR 322) could be obtained by electroporation ofK. oxytoca cells prepared at the OD₆₀₀ of 0.2 with 1.25 μg DNA at the filed strength of 2.5 kV, the parallel resistance of 200 Ω and capacitance of 25 μF.     

High frequency transformation of the industrial erythromycin-producing bacterium Saccharopolyspora erythraea

The DNA transformation in the industrial erythromycin-producing Saccharopolyspora erythraea was investigated as standard protoplast transformation methods are ineffective. Intergeneric conjugal transfer of DNA from E. coli demonstrated transformation efficiencies from 0.05 × 10⁻⁸ to 7.2 × 10⁻⁸ exconjugants generated per recipient. Electroporation-mediated methodologies were also established. More than 10⁵ transformants were acquired per μg DNA. The proposed protocol provides an alternative route for the introduction of DNA into industrial strains.     

Transformation ofLeuconostoc oenos by electroporation

Summary An electroporation-induced transformation system was developed forLeuconostoc oenos. Of the three plasmids tested (pGK13, pAMS100 and pIL253), only pGK13 was expressed. Several parameters were studied with possible influence on transformation efficiency. Electroporation conditions of 2000 V/cm, 25μF and 200 ohm proved successful. Transformation efficiencies (number of cells/μg plasmid DNA) of 1 X 10³ were obtained.     

A comparative study on the transformation of Aspergillus nidulans by microprojectile bombardment of conidia and a more conventional procedure using protoplasts treated with polyethyleneglycol

 An Aspergillus nidulans strain, auxotrophic for pyrimidine, was transformed to prototrophy by means of microprojectile bombardment. The transformation frequency was somewhat lower than conventional polyethyleneglycol-mediated transformation of protoplasts. However, the percentage of stable transformants was considerably higher with the biolistic approach. Typically, integrations of several copies of the plasmid introduced into chromosomal DNA were observed. The effect of several parameters, like the concentration of conidia, chamber pressure during bombardment and size of microprojectiles, on transformation frequencies were investigated and compared to previously published data on microprojectile bombardment of fungal conidia. Optimum results (6 transformants/μg plasmid DNA) were obtained when 10⁸ conidia were bombarded with a helium pressure of 5.5–8.3 MPa (800–1200 lb/in²). M5, M10 and M17 tungsten particles were equally efficient. Received: 9 August 1995/Received revision: 27 September 1995/Accepted: 4 October 1995     

Electroporation and conjugal plasmid transfer to members of the genus Aquaspirillum

Electroporation methods and conjugal matings were used to transfer several plasmid vectors to Aquaspirillum dispar and Aquaspirillum itersonii. The incompatibility P class plasmid RP4 was conjugally transferred from Escherichia coli HB101 to these spirilla, and the transconjugants subsequently donated the molecule to plasmid-free E. coli and A. dispar strains via conjugal matings. High-voltage electrotransformation was used to transfer plasmids pUCD2, pSa151 and RP4 to A. dispar and A. itersonii, at efficiencies as high as 3×10⁴ transformants per μg plasmid DNA. RP4 DNA isolated from spirillum hosts, but not RP4 from E. coli cells was successfully transferred to A. dispar and A. itersonii by electrotransformation, suggesting that modification and/or restriction activity may be present in these Aquaspirillum species.     

Electrotransformation of Chlorella vulgaris

Using hygromycin B resistance as a marker for selection, we have established the conditions required for the transformation of Chlorella vulgaris. The exponentially grown C. vulgaris cells were transformed by electroporation with plasmid pIG121-Hm, and transformants were selected with hygromycin B at a concentration of 50 μg/ml. Cell extracts prepared from the late-log cultures of the transformants exhibited glucuronidase activities as conferred by the gus gene on pIG121-Hm. The maintenance of plasmid in the algal cells seemed to be transient as many cultures derived from the hygromycin B-resistant colonies gradually lost the hygromycin resistance upon prolonged growth. The result of Southern blotting of the genomic DNAs prepared from transformant cultures exhibiting persistent hygromycin resistance showed that integration of part of the plasmid DNA into the host chromosome had taken place. Received: 19 December 1997 / Revision received: 5 October 1998 / Accepted: 27 October 1998     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Identification of a replicon from pCC3, a cryptic plasmid from <Emphasis Type="Italic">Leuconostoc citreum</Emphasis> C4 derived from kimchi,and development of a new host–vector system

Analysis of the structural properties of pCC3, a cryptic plasmid from Leuconostoc citreum C4 isolated from kimchi, determined its length as 3,338 bp and revealed three open reading frames (ORFs): ORF1–ORF3. ORF3 showed high homology with a replication initiation protein of the theta-type plasmid pTXL1. The fragment encompassing ORF3 and its upstream sequences (nt 1,299–1,634) was found to contain a functional plasmid replicon. A new shuttle vector, pUCC3E1, was constructed based on pCC3. Using Southern hybridization analysis, no single-stranded DNA intermediate was detected from Leu. citreum harboring pUCC3E1, which indicates that pCC3 replicated via the theta mechanism. The pUCC3E1 could be replicated in E. coli TG1 (5.8 × 10⁴ CFU/μg DNA) and the developed cloning hosts, Leu. citreum C16 (2.1 × 10² CFU/μg DNA) and Leu. citreum GJ7 (8.0 × 10¹ CFU/μg DNA). pUCC3E1 was stably maintained in Leu. citreum C16 (for 100 generations, ca. 94.2%) in the absence of erythromycin (5 μg/ml).     

Development of an electro-transformation system for Escherichia coli DH10B

Summary Escherichia coli can be transformed to high efficiencies by subjecting a mixture of cells and DNA to a brief but intense electrical field. Factors that affect the transformation efficiency of E.coli strain DH10B were analysed. Optimal conditions gave an efficiency of 10⁸ to 10⁹ transformants/g DNA with E.coli strains K803 and DH10B, and plasmids pB1221.23 and pBSK+. The use of ligated DNA resulted in 10⁶ transformants/g DNA. Detailed protocols for these systems are given.     

The influence of the growth phase of enteric bacteria on electrotransformation with plasmid DNA

Salmonella typhimurium LB5000 andEscherichia coli JM109 were transformed by electroporation. In accordance with the chemical transformation methods, the growth phase of these electrocompetent bacteria had a strong impact on transformation efficiency. Survival of bacteria, after the high-voltage electrical pulse was also influenced by the growth phase. Both bacterial species were most successfully electrotransformed when microbial cells were harvested at the late lag phase. The second optimum for transformation reachedE. coli cells in the mid-exponential andS. typhimurium cells in the late exponential phase. Transformation efficiencies ranged from 3.4×10⁴ to 2.7×10⁵ transformants per μg DNA in the case ofS. typhimurium and from 2.8 × 10² to 8.8×10⁵ transformants per μg DNA in the case ofE. coli. Survival of cells after the electrical pulse in late lag and late exponential phases was about 20% higher than during other phases of growth. Preparing electrocompetent cells from later phases of their growth is more useful for practice, because it provides more biomass with good yield of transformants.     

Genetic introduction of foreign genes to <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> by restriction enzyme-mediated integration

Pleurotus eryngii was transformed via restriction enzyme-mediated integration. In order to construct the transformation plasmid, the enhanced cyan fluorescent protein (ECFP) gene was ligated next to the gpd promoter of the plasmid pAN7-1. Transformation was facilitated via the heat treatment of a transformation mixture containing 1 μg of the HindIII-digested plasmid DNA and 10⁶ mushroom protoplasts in 40% polyethyleneglycol solution, resulting in 10–40 hygromycin-resistant transformants. Successful transformation was evidenced by PCR, Southern blot, and confocal fluorescence microscopic analyses on the selected transformants. To date, this is the first report on the transformation of P. eryngii by REMI technique.     

Electroporation and stable maintenance of plasmid DNAs in a biocontrol strain of Pseudomonas syringae

Transformation efficiencies as high as 10⁷ transformants g^–1 DNA have been previously reported for pseudomonads using electroporation protocols established for E. coli with plasmid DNAs prepared from methylation proficient E. coli hosts. We report here a protocol for electroporation of plasmid DNAs into a biocontrol strain of Pseudomonas syringae which could not be electroporated by standard E. coli methods. Transformation efficiencies of 10⁷ or higher were obtained with DNA recovered from initial P. syringae transformation or with DNA prepared from methylation deficient E. coli. Both plasmids used in this study were stably maintained in the absence of selection for at least 50 generations.     

Identification,cloning, and functional characterization of EmrD-3, a putative multidrug efflux pump of the major facilitator superfamily from <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O395

A putative multidrug efflux pump, EmrD-3, belonging to the major facilitator superfamily (MFS) of transporters and sharing homology with the Bcr/CflA subfamily, was identified in Vibrio cholerae O395. We cloned the emrD-3 gene and evaluated its role in antimicrobial efflux in a hypersensitive Escherichia coli strain. The efflux activity of this membrane protein resulted in lowering the intracellular concentration of ethidium. The recombinant plasmid carrying emrD-3 conferred enhanced resistance to several antimicrobials. Among the antimicrobials tested, the highest relative increase in minimum inhibitory concentration (MIC) of 102-fold was observed for linezolid (MIC = 256 μg/ml), followed by an 80.1-fold increase for tetraphenylphosphonium chloride (TPCL) (156.2 μg/ml), 62.5-fold for rifampin (MIC = 50 μg/ml), >30-fold for erythromycin (MIC = 50 μg/ml) and minocycline (MIC = 2 μg/ml), 20-fold for trimethoprim (MIC = 0.12 μg/ml), and 18.7-fold for chloramphenicol (MIC = 18.7 μg/ml). Among the fluorescent DNA-binding dyes, the highest relative increase in MIC of 41.7-fold was observed for ethidium bromide (125 μg/ml) followed by a 17.2-fold increase for rhodamine 6G (100 μg/ml). Thus, we demonstrate that EmrD-3 is a multidrug efflux pump of V. cholerae, the homologues of which are present in several Vibrio spp., some members of Enterobacteriaceae family, and Gram-positive Bacillus spp.     

High-Efficiency Transformation of Rhizobium leguminosarum by Electroporation

Electrotransformation of Rhizobium leguminosarum was successfully carried out with a 15.1-kb plasmid, pMP154 (Cm^r), containing a nodABC-lacZ fusion by electroporation. The maximum transformation efficiency, 10⁸ transformants/μg of DNA, was achieved at a field strength of 14 kV/cm with a pulse of 7.3 ms (186 Ω). The number of transformants was found to increase with increasing cell density, with no sign of saturation. In relation to DNA dosage, the maximum transformation efficiency (5.8 × 10⁸ transformants/μg of DNA) was obtained with 0.5 μg of DNA/ml of cell suspension, and a further increase in the DNA concentration resulted in a decline in transformation efficiency.