Transformation of Azotobacter vinelandii OP with a broad host range plasmid containing a cloned chromosomal nif-DNA marker

The non-nitrogen-fixing (Nif-) strain UW10 of Azotobacter vinelandii OP (UW) was naturally induced to competence and transformed with broad host range plasmid pKT210 containing the cloned wild-type nif-10 locus from A. vinelandii UW (Nif+); this marker was unable to complement the nif-10 mutation in trans, but could through recombination with the chromosome. The most frequent type of transformation event observed was recombination between the homologous regions of the plasmid and chromosome (producing Nif+ transformants) with loss of the plasmid vector. At a substantially lower frequency, transformants expressing the plasmid-encoded antibiotic resistance determinants were isolated which were phenotypically Nif-. Agarose gel electrophoresis showed that these transformants contained a plasmid migrating with the same mobility as the original donor plasmid. During culture these transformants acquired a Nif+ phenotype without the loss of the plasmid, as judged by the use of a hybridization probe specific for the cloned nif-DNA fragment. These data indicate that plasmids carrying sequences homologous to chromosomal sequences could be maintained in recombination-proficient A. vinelandii UW. The introduction of plasmids containing sequences homologous to chromosomal sequences was facilitated by prelinearization of the plasmid using a restriction endonuclease generating cohesive ends. Because the site of linearization could be chosen outside the region of shared homology, it was unlikely that the route of plasmid establishment occurred via a homology-facilitated transformation mechanism. The data also indicated that A. vinelandii UW could harbor broad host range cloning vectors based on plasmid RSF1010 without significant impairment of its nitrogen-fixation ability.     

Transformation of Azotobacter vinelandii with plasmid DNA.

Azotobacter vinelandii cells can be transformed at high frequencies with the broad-host-range plasmids pRK2501, RSF1010, and pGSS15, using a modification of the procedure developed by Page and von Tigerstrom (J. Bacteriol. 139:1058-1061, 1979) for chromosomal DNA-mediated transformation. The frequency of transformation per microgram of plasmid DNA per viable cell with pRK2501 and pGSS15 was about 5 X 10(-2) and 2 X 10(-2), respectively. With RSF1010, transformation frequencies ranged from 3 X 10(-4) to 4 X 10(-2). With each plasmid, the frequency of transformation was independent of the phase of the growth cycle. When concentrations of pRK2501 ranging from 0.1 to 51 micrograms of DNA were tested, the frequency of transformation was directly proportional to the amount of DNA. This linear response indicated that, although the uptake of plasmid DNA with this procedure may be inefficient, there is a high probability that once inside a cell the plasmid will be stably maintained. Cells that have been transformed with pRK2501 did not grow well on transforming medium which lacks iron and contains fixed nitrogen. However, on growth medium which contains iron and lacks fixed nitrogen, transformants produced distinctive colonies larger than those of nontransformed cells. Resistance to kanamycin due to transformation by pRK2501 was stably maintained for at least 10 successive generations in the absence of selective pressure. The present protocol should facilitate the molecular cloning of genes in Azotobacter spp.     

Characteristics and transformation of Zymomonas mobilis with plasmid pKT230 by electroporation

Transformation of Zymomonas mobilis with plasmid pKT230 by electroporation was achieved with a transformation efficiency of 9.0?±?1.8?×?10³ per μg plasmid DNA. The growing state of the host cells before transformation, the RC time constant for pulsing at the optimal electric field strength (7.5?kV/cm), the plasmid concentration and the post-incubation time prior to outgrowth in RM medium were the sensitive factors influencing the efficiency of the transformation. The data from batch cultures revealed that the plasmid-harboring cells, Z. mobilis (pKT230), had the same growth pattern as plasmid-free cells. The yield factors of biomass production and ethanol formation by Z. mobilis were nearly unchanged after being transformed and grown in the selective medium where the gene for antibiotic resistance was expressed. The results suggested that the plasmid pKT230 was stable in Z. mobilis and qualified for being a cloning vector in the construction of a recombinant ethanol-producer.     

A plasmid which can be transferred between Escherichia coli and Pasteurella haemolytica by electroporation and conjugation

Three broad-host-range plasmids (pRK290, pSa4 and pKT230) and one native Pasteurella haemolytica plasmid (pPH33) were used in transformation experiments with P. haemolytica strains T179 (serotype A1), Y216 (serotype A2) and its capsular-deficient variant Y216/NS1. No transformants were detected with either heat-shock or freeze-thaw techniques. However, by electroporation, all P. haemolytica strains were transformed by pPH33 but not by pRK290 or pSa4. The highest frequency obtained was 91 x 10(4) transformants per microgram of pPH33 DNA with P. haemolytica strain Y216/NS1. Although pPH33 itself was non-transmissible by conjugation, it could be mobilized from Escherichia coli, using the transfer function of the IncP plasmid pRK2013, into P. haemolytica at a frequency of 0.3-2.2 x 10(-3) per recipient cell.     

Plasmid transformation of naturally competent Acinetobacter calcoaceticus in non-sterile soil extract and groundwater

The natural transformation of Acinetobacter calcoaceticus BD413 (trp E27) was characterized with respect to features that might be important for a possible gene transfer by extracellular DNA in natural environments. Transformation of competent cells with chromosomal DNA (marker trp ⁺) occurred in aqueous solutions of single divalent cations. Uptake of DNA into the DNase I-resistant state but not the binding of DNA to cells was strongly stimulated by divalent cations. An increase of transformation of nearly 3 orders of magnitude was obtained as a response to the presence of 0.25 mM Ca²⁺. With CaCl₂ solutions the transformation frequencies approached the highest values obtained under standard broth conditions, followed by MnCl₂ and MgCl₂. It is concluded that transformation requires divalent cations. DNA competition experiments showed that A. calcoaceticus does not discriminate between homologous and heterologous DNA. Furthermore, circular plasmid DNA competed with chromosomal DNA fragments and vice versa. The equally efficient transformation with plasmid pKT210 isolated from A. calcoaceticus or Escherichia coli indicated absence of DNA restriction in transformation. High efficiency plasmid transformation was obtained in samples of non-sterile natural groundwater and in non-sterile extracts of fresh and air-dried soil. Heat-treatment (10 min, 80°C) of the non-sterile liquid samples increased transformation only in the dried soil extract, probably by inactivation of DNases. The results presented suggest that competent cells of A. calcoaceticus can take up free high molecular weight DNA including plasmids of any source in natural environments such as soil, sediment or groundwater.     

Characterization of genetic transformation in Streptococcus mutans by using a novel high-efficiency plasmid marker rescue system.

We developed a marker rescue system for study of competence development and genetic transformation in Streptococcus mutans. The system involved the recombinational rescue of a tetracycline resistance (Tcr) determinant by a homologous, inactive locus (Tcs because of a small deletion). Streptococcal cells harboring this in vitro-prepared Tcs construct (pVA1208) were restored to Tcr when plasmid (pVA981) DNA was used as donor material. pVA981 contained the intact streptococcal Tcr locus and was unable to autonomously replicate in streptococci. Marker rescue with this system followed first-order kinetics and occurred at a frequency 8- or 160-fold higher than did transformation with homologous chromosomal or plasmid DNA, respectively. By using the rescue system, we were able to confirm that competence of S. mutans appeared to be inducible. This was indicated by a sequential increase and then decrease in Tcr transformation frequencies during growth in complex medium. Also, donor DNA binding was not sequence specific, since the recovery of Tcr transformants was reduced by increasing the concentrations of heterologous DNA. We investigated the fate of donor DNA and the kinetics of plasmid establishment in the transformation of S. mutans with plasmid DNA. Monomeric plasmid molecules transformed S. mutans as a second-order process, whereas multimeric plasmid DNA and chromosomal markers were recovered as a first-order process. Approximately 50% of the initially bound donor plasmid DNA was found to remain in a trichloroacetic acid-insoluble form. Our results suggested that molecular cloning in S. mutans would be conducted most efficiently by using helper plasmid systems or shuttle vectors and that gene transfer by transformation of S. mutans occurred in a manner similar to that observed in Streptococcus sanguis.     

Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens

A reliable and efficient method has been developed for the electroporation-mediated transformation of Clostridium perfringens with plasmid DNA. Transformation of vegetative cells of C. perfringens strain 13 with the 7.9-kb Escherichia coli-C. perfringens shuttle plasmid pHR 106 required pretreatment with lysostaphin (2 to 20 micrograms/ml) for 1 h at 37 degrees C. Cells harvested early in the logarithmic stage of growth were transformed more efficiently than cells at other growth phases. The transformation frequency increased with the DNA concentration, to a saturating level at 5 to 10 micrograms DNA/ml. The transformation frequency was proportional to the field strength and time constant of the electroporation pulse; however, the field strength was a far more important parameter. A cell density between 1 x 10(8) and 5 x 10(8) cells/ml proved to be optimal for transformation. The procedure was capable of generating up to 3.0 x 10(5) transformants per micrograms DNA. The potential value of the method for the cloning of C. perfringens genes was demonstrated by the cloning of the clostridial tetracycline-resistance determinant, tetP, from the E. coli recombinant plasmid pJIR71, into C. perfringens strain 13.     

Cloning of tryptophanase gene of Alcaligenes faecalis for effective production of l-tryptophan

For the effective production of l-tryptophan from indole and l-serine by the action of tryptophanase from Alcaligenes faecalis, cloning of the enzyme gene (tna) was studied. A. faecalis was transformed not only by broad host range plasmid pKT231 but also by pLG338, pACYC177, and pBR322 derivatives. A recombinant tna plasmid was isolated by shotgun experiments with Escherichia coli K-12, and the isolated tna gene located on the 3.2 kb DNA fragment. Tryptophanase activity of A. faecalis transformed by the tna recombinant plasmid was about 4-fold higher than that of wild cells.     

Pseudomonas stutzeri and related species undergo natural transformation

Cells of Pseudomonas stutzeri are naturally transformed by homologous chromosomal DNA; they do not require chemical treatment to become competent. This capacity to undergo natural transformation was found to be shared by the closely related species P. mendocina, P. alcaligenes, and P. pseudoalcaligenes, but was not detectable in strains of P. aeruginosa, P. perfectomarinus, P. putida, P. fluorescens, or P. syringae. P. stutzeri could be transformed either on plates or in liquid medium. Only double-stranded chromosomal DNA was effective; single-stranded DNA and plasmid DNA were not. DNA fragments larger than 10 kilobase pairs were more effective than smaller fragments. The transformation frequency was proportional to DNA concentration from 1 ng/ml to 1 microgram/ml; higher concentrations were saturating. The maximum frequency, about 10(-4) transformants per recipient cell, was obtained with cells from a culture in the early stationary growth phase. A variety of chromosomal mutations have been transformed, including mutations to auxotrophy and to antibiotic resistance. Other systems for genetic exchange in P. stutzeri have not yet been found; transformation offers a means for the genetic analysis of this metabolically versatile organism.     

Transformation ofStreptomyces avermitilis by plasmid DNA

Summary Polyethylene glycol (PEG) efficiently mediated the transformation ofStreptomyces avermitilis protoplasts by plasmid DNA to yield 10⁷ transformants per g of plasmid DNA. Under conditios in which the maximum transformation frequency was observed, the cotransformation frequency exceeded 10%. The number of transformants increased linearly with the amount of DNA and number ofS. avermitilis protoplasts. Relaxed and supercoiled, but not linear DNA transformed protoplasts efficiently. Dimethyl sulfoxide (DMSO)-mediated transformation of protoplasts was 1000-fold less efficient. PEG and, less efficiently, DMSO also mediated the transformation of whole cells ofS. avermitilis by DNA.     

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.     

Stable transformation of the cyanobacterium Synechocystis sp. PCC 6803 induced by UV irradiation.

Irradiation of the photoheterotrophic cyanobacterium Synechocystis sp. PCC 6803 with low levels of UV light allows for stable, integrative transformation of these cells by heterologous DNA. In this system, transformation does not rely on an autonomously replicating plasmid and is independent of homologous recombination. Cells treated with UV light in the absence of DNA and cells given DNA but not exposed to UV do not yield antibiotic-resistant colonies in platings of up to 2 X 10(8) cells. Optimal conditions for this UV-induced transformation are described. Analysis of the transformants indicates that (i) only a segment of the introduced plasmid is found in the DNA of the transformed cells; (ii) in independently isolated clones, DNA insertion apparently occurs at different sites in the chromosome; and (iii) hybridization data suggest that insertion in one of the transformants may have occurred into a region of the chromosome that is repeated or that integration of plasmid DNA may have been accompanied by a rearrangement or duplication of DNA sequences near the insertion site. DNA isolated from the primary transformants as well as a cloned fragment containing the UV-inserted plasmid sequence and flanking cyanobacterial DNA transform wild-type cells at a high frequency (5.0 X 10(-4) and 1.5 X 10(-5), respectively). Possible mechanisms of this transformation system are discussed, as are the potential uses of this system as an integrative cloning-complementation vector and as a mutagenic agent in which the genetic lesion is already tagged with a selectable marker.     

Transformation and allelic replacement in Francisella spp.

We describe methods for transposon mutagenesis and allelic replacement in the facultative intracellular pathogen Francisella. Recombinant clones were constructed by insertion of partially cut F. tularensis or F. novicida DNA into pUC19 and then mutagenized with a mini-Tn10-Km transposon. F. novicida could be transformed with these plasmids either by a chemical transformation method or by electroporation, whereas F. tularensis could be transformed only by electroporation. Transformation of F. tularensis by electroporation was enhanced in the absence of the capsule. Southern blot analysis showed that the KmR marker was rescued either by integration of the plasmid into the Francisella chromosome or by allelic replacement. Allelic replacement was found to be the mechanism underlying a site-specific mutation affecting FopA, an outer-membrane protein of Francisella. F. novicida could also be transformed with chromosomal DNA carrying the KmR marker and the transformation frequency obtained using chromosomal DNA was generally greater than that obtained using plasmid DNA. F. novicida was also transformed by an IncQ plasmid containing an F. novicida DNA insert, which replicated autonomously in this host.     

Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors

We asked if single-stranded vector DNA molecules could be used to reintroduce cloned DNA sequences into a eukaryotic cell and cause genetic transformation typical of that observed using double-stranded DNA vectors. DNA was presented to Saccharomyces cerevisiae following a standard transformation protocol, genetic transformants were isolated, and the physical state of the transforming DNA sequence was determined. We found that single-stranded DNA molecules transformed yeast cells 10- to 30-fold more efficiently than double-stranded molecules of identical sequence. More cells were competent for transformation by the single-stranded molecules. Single-stranded circular (ssc) DNA molecules carrying the yeast 2 μ plasmid-replicator sequence were converted to autonomously replicating double-stranded circular (dsc) molecules, suggesting their efficient utilization as templates for DNA synthesis in the cell. Single-stranded DNA molecules carrying 2 μ plasmid non-replicator sequences recombined with the endogenous multicopy 2 μ plasmid DNA. This recombination yielded either the simple molecular adduct expected from homologous recombination (40% of the transformants examined) or aberrant recombination products carrying incomplete transforming DNA sequences, endogenous 2 μ plasmid DNA sequences, or both (60% of the transformants examined). These aberrant recombination products suggest the frequent use of a recombination pathway that trims one or both of the substrate DNA molecules. Similar aberrant recombination products were detected in 30% of the transformants in cotransformation experiments employing single-stranded and double-stranded DNA molecules, one carrying the 2 μ plasmid replicator sequence and the other the selectable genetic marker. We conclude that single-stranded DNA molecules are useful vectors for the genetic transformation of a eukaryotic cell. They offer the advantage of high transformation efficiency, and yield the same intracellular DNA species obtained upon transformation with double-stranded DNA molecules. In addition, single-stranded DNA molecules can participate in a recombination pathway that trims one or both DNA recombination substrates, a pathway not detected, at least at the same frequency, when transforming with double-stranded DNA molecules     

High frequency vector-mediated transformation and gene replacement in Tetrahymena.

Recently, we developed a mass DNA-mediated transformation technique for the ciliated protozoan Tetrahymena thermophila that introduces transforming DNA by electroporation into conjugating cells. Other studies demonstrated that a neomycin resistance gene flanked by Tetrahymena H4-I gene regulatory sequences transformed Tetrahymena by homologous recombination within the H4-I locus when microinjected into the macronucleus. We describe the use of conjugant electrotransformation (CET) for gene replacement and for the development of new independently replicating vectors and a gene cassette that can be used as a selectable marker in gene knockout experiments. Using CET, the neomycin resistance gene flanked by H4-I sequences transformed Tetrahymena, resulting in the replacement of the H4-I gene or integrative recombination of the H4-I/neo/H4-I gene (but not vector sequences) in the 5' or 3' flanking region of the H4-I locus. Gene replacement was obtained with non-digested plasmid DNA but releasing the insert increased the frequency of replacement events about 6-fold. The efficiency of transformation by the H4-I/neo/H4-I selectable marker was unchanged when a single copy of the Tetrahymena rDNA replication origin was included on the transforming plasmid. However, the efficiency of transformation using CET increased greatly when a tandem repeat of the replication origin fragment was used. This high frequency of transformation enabled mapping of the region required for H4-I promoter function to within 333 bp upstream of the initiator ATG. Similarly approximately 300 bp of sequence downstream of the translation terminator TGA of the beta-tubulin 2 (BTU2) gene could substitute for the 3' region of the H4-I gene. This hybrid H4-I/neo/BTU2 gene did not transform Tetrahymena when subcloned on a plasmid lacking an origin of replication, but did transform at high frequency on a two origin plasmid. Thus, the H4-I/neo/BTU2 cassette is a selectable marker that can be used for gene knockout in Tetrahymena. As a first step toward constructing a vector suitable for cloning genes by complementation of mutations in Tetrahymena, we also demonstrated that the vector containing 2 origins and the H4-I/neo/BTU2 cassette can co-express a gene encoding a cycloheximide resistant ribosomal protein.     

Natural transformation of a marineVibrio species by plasmid DNA

Vibrio sp. DI9, recently isolated from Tampa Bay, FL, has been found to be naturally transformed by the broad host range plasmid pKT230 in both filter transformation assays and sterile sediment microcosms. This is the first report of natural transformation by plasmid DNA of aVibrio sp. and of a marine bacterial isolate. Transformation frequencies ranged from 0.3 to 3.1×10^–8 transformants per recipient. Transformants were detected by both plating and by selection for growth in liquid medium in the presence of streptomycin and kanamycin and confirmed by probing of southern transfers. Transformation was enhanced by multimeric forms of the plasmid. A technique using sediment microcosms, mixed populations ofVibrio sp. DI9 and another antibiotic resistant organism, and enrichment in liquid media has been developed which allows detection of transformation at frequencies too low to be detected by plating. This technique may serve as a model for the detection of natural transformation in the environment. These results suggest that natural transformation may be one mechanism of horizontal plasmid transfer in the marine environment, and may provide the methodology with which to detect this process in natural populations of bacteria.     

Efficient transformation of Dictyostelium discoideum amoebae.

We have transformed Dictyostelium discoideum amoebae by using derivatives of a plasmid, pAG60, which was designed for transformation of mammalian cells. The plasmid carries the promoter region of the herpes simplex virus type 1 thymidine kinase gene linked to the bacterial gene kan, which codes for the enzyme aminoglycoside 3'-phosphotransferase. kan is derived from the Tn5 transposon. Expression of the phosphotransferase permits direct selection of transformed cells by their resistance to the antibiotic G-418. pAG60 is incapable of transforming D. discoideum but is made transformation proficient by cloning D. discoideum sequences into the tetracycline resistance gene. The majority of transformed cells grow and develop normally and differentiate to give G-418-resistant spores. These transformants are unstable and rapidly lose their G-418-resistance during growth in the absence of antibiotic selection. Southern blots show that these unstable G-418-resistant transformants carry the pBR322 and kan sequences of pAG60. The pAG60-D. discoideum recombinant plasmids used for transformation were constructed in a way that might make them mutagenic. We have isolated several developmental mutants after transformation of D. discoideum with libraries of pAG60-D. discoideum recombinant plasmids. These mutants are G-418 resistant and carry pAG60 in their nuclear DNA. We recovered a pAG60-D. discoideum recombinant plasmid from several developmental mutants. This plasmid transforms D. discoideum at an elevated frequency and integrates into the nuclear genome. We speculate that integration can result in insertional inactivation of genes that are essential for differentiation but not for growth. Mutagenic transformation occurred only if the transforming plasmid had homology with D. discoideum nuclear DNA. A mammalian cell transformation vector, pSV2-neo, carried no D. discoideum sequences and was able to transform. However, pSV2-neo transformation was not mutagenic. These results suggest that direct inactivation and recovery of genes that are essential for differentiation of D. discoideum will be possible.     

Natural transformation in Campylobacter species.

Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without the requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 10(-3) transformants per recipient cell in C. coli and 10(-4) in C. jejuni. Maximum competence was found in the early log phase of growth. Campylobacters preferentially took up their own DNA in comparison with Escherichia coli chromosomal DNA, which was taken up very poorly. Three new Campylobacter spp.-to-E. coli shuttle plasmids, which contained additional cloning sites and selectable markers, were constructed from the shuttle vector pILL550A. These plasmid DNAs were taken up by campylobacters much less efficiently than was homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 10(-4) transformants per cell were obtained. The tetM determinant, originally obtained from Streptococcus spp. and not heretofore reported in Campylobacter spp., was isolated from an E. coli plasmid and was introduced, selecting for tetracycline resistance, by natural transformation into C. coli.