Transformation of Streptococcus sanguis (Challis) by linear plasmid molecules

Summary The streptococcal erythromycin resistance plasmid pSM9 was used to study the problem of how the transforming activity of mixtures of two unique linear products of restriction enzyme digestion depends on the distance between the cleavage sites. In transformation of the Challis strain of S. sanguis, the transforming activity of mixed digests increased with increasing relative distances (x) between the restriction sites, where 0x0.5. To explain the experimental results, a mathematical model was proposed according to which the overall probability (p) of transformation resulting in a functional replicon is the product of the partial probabilities of initial single-strand pairing, circularization, and stability of the paired intermediate, all of which were assumed to depend on x. A linear relationship found between transformation frequency and p was taken to support the model. Transformation of Challis by mixtures of two linearized plasmid molecules with regions of internal nonhomology resulting in paired intermediates with insertion or substitution loops allowed either donor molecule to contribute to the transformation yield.     

Efficient gene targeting in the filamentous fungusAlternaria alternata

To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1^–) transformants accounted for 30 to 80% of hygromycin B-resistant (Hy^R) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1^– transformants accounted for only 5% of Hy^R transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1^–. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.     

Linear DNA must have free ends to transform rat cells efficiently

Summary We have observed that failure to remove certain restriction enzymes after digestion reduced the transforming ability of DNA from 10- to 50-fold. The DNA found integrated in the transformed cells isolated under these conditions had lost little or no sequences. We interpret these results as indicating that certain restriction enzymes remain bound to the DNA ends after digestion, thus generating a substrate unfavorable both for integration and exonucleolytic degradation. As expected from this interpretation, removal of the restriction enzymes before transfection restored the full transforming ability of linear DNA, but also resulted in the integrated sequences being significantly shorter than the transfected DNA. These findings strongly argue for the hypothesis that integration of linear DNA by illegitimate recombination requires free ends and further suggest that exonucleolytic degradation of such ends may generate a preferred substrate for integration. Finally, a comparison of the sequences found integrated after transfection with circular or linear molecules, led us to conclude that circular molecules need not be linearized to become integrated.     

Linearization of donor DNA during plasmid transformation in Neisseria gonorrhoeae.

We examined the fate of plasmid DNA after uptake during transformation in Neisseria gonorrhoeae. An 11.5-kilobase plasmid, pFA10, was processed to linear double-stranded DNA during uptake by competent cells, but cleavage of pFA10 was not site specific. A minority of pFA10 entered as open circles. A 42-kilobase plasmid, pFA14, was degraded into small fragments during uptake; no intracellular circular forms of pFA14 were evident. Since pFA10 DNA linearized by a restriction enzyme was not further cut during uptake, the endonucleolytic activity associated with entry of plasmid DNA appeared to act preferentially on circular DNA. Although linear plasmid DNA was taken up into a DNase-resistant state as efficiently as circular DNA, linear plasmid DNA transformed much less efficiently than circular plasmid DNA. These data suggest that during entry transforming plasmid DNA often is processed to double-stranded linear molecules; transformants may arise when some molecules are repaired to form circles. Occasional molecules which enter as intact circles may also lead to transformants.     

Linear DNA introduced into carrot protoplasts by electroporation undergoes ligation and recircularization

The integrated DNA in stable transformants formed by direct gene transfer often shows complex restriction patterns. One cause of these complex restriction patterns could be the ligation of plasmid fragments prior to their integration. This paper provides evidence for the ligation of plasmid fragments by plant cells. Carrot protoplasts were electroporated in the presence of pCaMVCATM and assayed for chloramphenicol actyltransferase (CAT) activity 24h later. Linear and supercoiled forms of pCaMVCATM supported similar levels of CAT expression. Surprisingly, digestion of the plasmid at a site between the CaMV 35S promoter and the CAT coding region reduced expression by only 40–50%. Electroporation carried out in the presence of isolated plasmid fragments suggested that this result was due to ligation of the linearized plasmid by the protoplasts. CAT expression was obtained with a mixture of isolated CaMV 35S promoter and the CAT coding region; neither fragment alone supported expression. Further evidence of ligation was provided by electroporation of protoplasts in the presence of a mixture of linearized pGEM and the 1.5-kbHind III fragment of pCaMVCATM. DNA isolated from nuclei of the protoplasts was used to transform competent cells ofEscherichia coli, and colonies were recovered that carried pGEM withHind III-CaMVCAT inserts. Electroporation of protoplasts in the presence of linear and supercoiled pGEM and use of DNA isolated from nuclei to transformE. coli yielded an estimate of the frequency of plasmid ligation. A maximum of only 4% of the input linear DNA was recovered as circular molecules. This result suggests the frequency of ligation is low, but examination of the plasmid DNA in the plant nuclei by electrophoresis indicates extensive degradation of the plasmid and preferential loss of the circular forms. Thus, the ligated plasmids may be converted to the linear form and hence rendered unrecoverable by cloning intoE. coli.     

Autonomous replication of foreign DNA in Histoplasma capsulatum: role of native telomeric sequences.

Genetic transformation of the dimorphic pathogenic fungus Histoplasma capsulatum can result in chromosomal integration of the transforming DNA or the generation of multicopy linear plasmids carrying the transforming DNA. We showed previously that Escherichia coli plasmids do not replicate autonomously in H. capsulatum without significant modifications, one of which is the in vivo addition of Histoplasma telomeres at the termini of linear DNA. To address the requirements for autonomous replication in H. capsulatum, we constructed a circular E. coli plasmid containing adjacent inverted stretches of Histoplasma telomeric repeats separated by a unique restriction site. The linearized plasmid bearing telomeric termini was maintained in H. capsulatum without modification other than the addition of more telomeric sequence. We recovered the original plasmid in E. coli after removal of the telomeric termini by using engineered restriction sites. Thus, no special Histoplasma modification or sequence other than the telomeres was needed for autonomous replication in H. capsulatum. Additionally, this plasmid provides a shuttle vector that replicates autonomously in E. coli (as a circular plasmid) and in H. capsulatum (as a linear plasmid).     

Transformation of group F streptococci by plasmid DNA.

When the Challis strain of Streptococcus sanguis was transformed by the 17 megadalton beta plasmid from Streptococcus faecalis strain DS5, the plasmid underwent a 1.5 megadalton deletion (LeBlanc & Hassell, 1976). Furthermore, the covalently closed circular (CCC) plasmid DNA isolated from Challis transformants was rapidly converted to a linear form which did not possess any detectable transforming activity. To obtain stable CCC plasmid DNA a competent culture of a Lancefield group F streptococcus, strain DL8 (ATCC 12393), was used as a recipient of beta plasmid DNA. The plasmid DNA isolated from group F transformants exhibited the same configuration and size characteristics as the DS5 beta plasmid, and the CCC configuration was stable upon storage. CCC plasmid DNA from a group F transformant was biologically active and, when added to competent cultures of strain DL8, transformed them at frequencies about 100-fold greater than did beta plasmid DNA from DS5. This suggests the existence of a restriction--modification system in strain DL8.     

Intramolecular recombination during plasmid transformation of Bacillus subtilis competent cells.

We have constructed plasmids carrying direct internal repeats 260-2000 bp long. Monomers of such plasmids transformed Bacillus subtilis competent cells. The efficiency of transformation varied with the square of the length of repeats. The transformed clones harbored either the entire transforming plasmid and the plasmid arising by recombination between the repeats, or only the latter plasmid. Internally-repeated plasmids linearized by in vitro cleavage with restriction endonuclease could transform, yielding clones which exclusively harbored a plasmid resulting from recombination between the repeats. When the transforming plasmid carried repeats which differed slightly, conversion of one repeat into the other could occur. The following model of plasmid transformation accounts for these data: (1) plasmid DNA is cleaved and rendered linear in contact with competent cells; (2) a linear, at least partially double-stranded plasmid molecule is introduced or formed by repair within the cell; (3) a circular viable plasmid is produced by recombination between repeats carried on this molecule; (4) alternatively, a viable plasmid is produced by repairing the cut within one of the repeats by DNA synthesis which uses the other repeat as a template.     

Recombination between heterologous linear and circular mitochondrial plasmids in the fungus Neurospora

A strain of Neurospora intermedia from China contains five prominent extragenomic mitochondrial plasmids: three linear elements called zhisi plasmids, and two circular plasmids, Harbin-1 and -2. In one subculture, levels of four plasmids (all three zhisis and Harbin-1) fell to undetectable values and two novel linear plasmids appeared, Harbin-L and -L2, as well as a new small circular plasmid, Harbin-0.9. Cross-hybridization of restriction fragments and DNA sequencing showed that the Harbin-L plasmid was composed of parts of the circular Harbin-1 plasmid and of one of the linear zhisi plasmids. A model is presented in which the Harbin-1 and zhisi plasmids are present within the same mitochondrion, and crossovers at two separate 7 by sites of sequence identity effectively insert part of the circular Harbin-1 DNA into a zhisi linear plasmid, simultaneously deleting part of the zhisi element. The small plasmid Harbin-0.9 is a fragment of the Har-1 plasmid, and seems to be another product of the recombination process that created Har-L. Recombination of this type could have contributed to the wide array of mitochondrial plasmids found in natural populations of Neurospora.     

Efficient gene targeting in the filamentous fungusAlternaria alternata

To characterize homologous recombination of transforming DNA in the filamentous fungusAlternaria alternata, we have compared the frequencies of gene targeting by circular and linear DNA fragments in the fungus. TheA. alternata BRM1 gene, which is an essential gene for melanin biosynthesis, was selected as a target locus.BRM1 targeting events are easily identified because loss of function leads to a change in mycelial color from black to light brown. We constructed targeting vectors by inserting 0.6 to 3.1 kb internalBRM1 segments into a plasmid containing the hygromycin B phosphotransferase gene. When circular plasmids were used, melanin-deficient (Me1^?) transformants accounted for 30 to 80% of hygromycin B-resistant (Hy^R) transformants, correlating closely with the size of theBRM1 segment in the transforming DNA. Restriction enzyme digestion within theBRM1 region greatly enhanced the frequency of gene targeting: integration of the linear plasmids was almost completely attributable to homologous recombination, regardless of the size of theBRM1 segments. Plasmids carrying bothBRM1 segments and rDNA segments were transformed into the fungus to examine the effect of the number of target copies on homologous recombination. Using the circular plasmids, Me1^? transformants accounted for only 5% of Hy^R transformants. In contrast, when the linear plasmid produced by restriction enzyme digestion within theBRM1 segment was used, almost all transformants were Me1^?. These results indicate that homologous integration of circular molecules inA. alternata is sensitive to the length of homology and the number of targets, and that double-strand breaks in transforming DNA greatly enhance homologous recombination.     

Recombination following transformation of Escherichia coli by heteroduplex plasmid DNA molecules

Circular heteroduplex DNA molecules introduced into Escherichia coli-competent cells are converted to new recombinant plasmids as a result of enzymatic actions in vivo. A pair of plasmids with partial sequence homology were each linearized at a different position with restriction enzymes, and the termini were made flush with the single-strand-specific S1 nuclease. Duplex molecules were then formed by melting and annealing these plasmid DNAs together. In contrast to linear homoduplex molecules, heteroduplexes circularize and therefore transform E. coli efficiently. Unique DNA sequences on each of the parental strands in the transforming heteroduplexes can be selectively incorporated or deleted as a result of in vivo enzymatic activities in transformed cells. This method permits the generation of new recombinant sequences in vivo without relying solely on the presence of convenient restriction sites for manipulation of DNA fragments in vitro.     

Different specific activities of the monomeric and oligomeric forms of plasmid DNA in transformation of B. subtilis and E. coli.

Summary (1) The low residual transforming activity in preparations of monomeric, supercoiled, circular (CCC) forms of the plasmids pC194 and pHV14 could be attributed to the presence in such isolates of a small number of contaminating multimeric molecules. (2) E. coli derived preparations of pHV14, an in vitro recombinant plasmid capable of replication in both E. coli and B. subtilis, contain oligomeric forms of plasmid DNA in addition to the prevalent monomeric CCC form. The specific transforming activity of pHV14 DNA for E. coli is independent of the degree of oligomerization, whereas in transformation of B. subtilis the specific activity of the purified monomeric CCC molecules is at least four orders of magnitude less than that of the unfractionated preparation. (3) Oligomerization of linearized pHV14 DNA by T4 ligase results in a substantial increase of specific transforming activity when assayed with B. subtilis and causes a decrease when used to transform E. coli.     

Transformation of Streptococcus sanguis Challis by plasmid deoxyribonucleic acid from Streptococcus faecalis.

Plasmid deoxyribonucleic acid (DNA) from Streptococcus faecalis, strain DS5, was transferred to the Challis strain of Streptococcus sanguis by transformation. Two antibiotic resistance markers carried by the beta plasmid from strain DS5, erythromycin and lincomycin, were transferred to S. sanguis at a maximum frequency of 1.8 x 10-5/colony-forming unit. Approximately 70% of the covalently closed circular DNA isolated from transformant cultures by dye buoyant density gradients was shown to be hybridizable to beta plasmid DNA. Two major differences were observed between the beta plasmid from S. faecalis and the plasmid isolated from transformed S. sanguis: (i) the beta plasmid from strain DS5 sedimented in velocity gradients at 43S, whereas the covalently closed circular DNA from transformed Challis sedimented at 41S, suggesting a 1.5-Mdal deletion from the beta plasmid occurred; (ii) although the 43S beta plasmid remained in the supercoiled configuration for several weeks after isolation, the 41S plasmid was rapidly converted to a linear double-stranded molecule. Attempts to transform S. sanguis with the alpha plasmid from S. faecalis, strain DS5, were unsuccessful.     

Characterization of plasmid transformation in Bacillus subtilis: kinetic properties and the effect of DNA conformation.

Summary Transformation of competent cells of Bacillus subtilis with antibiotic resistance plasmid DNA has shown that (a) competence for plasmid and chromosomal DNA develops with similar kinetics; (b) DNA linearized with a variety of restriction endonucleases does not transform; (c) CCC plasmid DNA is inactivated for transformation by a single nick; (d) T4 ligase restores transforming activity to both nicked and linearized DNA; (e) CCC relaxed DNA is fully active in transformation; (f) the DNA concentration-dependence of plasmid transformation is first order; and (g) plasmid transformation proceeds with a low efficiency, requiring the uptake of 10³ to 10⁴ DNA molecules per transformant.Based on this information, a model for the processing of chromosomal, plasmid and transfecting DNA is proposed.Abbreviations Cm Chloramphenicol - Em erythromycin - Km kanamycin - Sm streptomycin - CCC covalently closed circular - TBAB tryptose blood agar base - NCE nicking and closing enzyme In partial fulfillment of the requirements for the doctoral degree in the Department of Microbiology at the New York University School of Medicine, for S.C.     

Selection for the transfer of phenotypically nonselectable chromosomal mutations to recombinant plasmids through introduction of an altered restriction site

We describe a simple method to select for transfer of mutant alleles from the Escherichia coli chromosome to a plasmid which formerly carried the wild-type (wt) allele. The wt allele on the plasmid is modified by introduction of a unique restriction site (e.g., XhoI) and transformed into a rec ⁺ strain carrying the mutant allele on the chromosome. Upon homogenotization, the efficiency of which was increased by UV irradiation of the transforming plasmid [Chattoraj et al., Gene 27 (1982) 213–222], plasmids carrying the mutant allele are formed which are resistant to XhoI. These plasmids are selected from the population by resistance to XhoI digestion coupled with the low transformation efficiency of linear DNA molecules in recA⁻ strain. The method is efficient and rapid and has particular advantages in situations where the mutant allele is difficult to detect by its phenotype.     

In vivo ligation of linear DNA molecules to circular forms in the yeast Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae was transformed with restriction endonuclease-digested (linear) DNAs containing the replication origin of the yeast 2 microns plasmid and selectable markers with efficiencies of 10(3) to 10(4), 10(3), and 10(2) to 10(3) transformants per microgram of DNA in the cases of transformations with linear DNAs containing the same cohesive ends, flush ends, and non-complementary cohesive ends, respectively. The results of a restriction analysis of the circular plasmids recovered from transformed cells suggested that the linear DNA molecules were ligated to produce circular forms in the recipient protoplasts.     

Recombination between heterologous linear and circular mitochondrial plasmids in the fungus Neurospora

A strain of Neurospora intermedia from China contains five prominent extragenomic mitochondrial plasmids: three linear elements called zhisi plasmids, and two circular plasmids, Harbin-1 and -2. In one subculture, levels of four plasmids (all three zhisis and Harbin-1) fell to undetectable values and two novel linear plasmids appeared, Harbin-L and -L2, as well as a new small circular plasmid, Harbin-0.9. Cross-hybridization of restriction fragments and DNA sequencing showed that the Harbin-L plasmid was composed of parts of the circular Harbin-1 plasmid and of one of the linear zhisi plasmids. A model is presented in which the Harbin-1 and zhisi plasmids are present within the same mitochondrion, and crossovers at two separate 7 by sites of sequence identity effectively insert part of the circular Harbin-1 DNA into a zhisi linear plasmid, simultaneously deleting part of the zhisi element. The small plasmid Harbin-0.9 is a fragment of the Har-1 plasmid, and seems to be another product of the recombination process that created Har-L. Recombination of this type could have contributed to the wide array of mitochondrial plasmids found in natural populations of Neurospora.     

Isolation of plasmids present in thermophilic strains from hot springs in Jordan

The plasmid profile of two thermophilic bacterial strains isolated from recreation thermal springs in Jordan has been investigated. These strains are Streptococcus thermophilus and Bacillus sp1, which have been isolated from Zerka – Maeen and Himma hot springs respectively. Supercoiled and circular plasmid forms were detected, explaining the effect of DNA conformation on the mobility of the plasmid in the agarose gel electrophoresis. Two plasmids have been isolated and characterized by restriction endonucleases to facilitate their use as cloning vectors in thermophilic strains. The sizes of the plasmids were approximately 3 kb (from Streptococcus thermophilus) and 7 kb (from Bacillus spl). These plasmids were then digested with three different restriction enzymes (EcoRI, Bam HI, and HindIII), one of which was found to possess a single site for both plasmids, this enzyme is EcoRI.     

Illegitimate integration of single-stranded DNA in Saccharomyces cerevisiae

 We studied illegitimate recombination by transforming yeast with a single-stranded (ss) non-replicative plasmid. Plasmid pCW12, containing the ARG4gene, was used for transformation of yeast strains deleted for the ARG4, either in native (circular) form or after linearization within the vector sequence by the restriction enzyme ScaI. Both circular and linearized ss plasmids were shown to be much more efficient in illegitimate integration than their double-stranded (ds) counterparts and more than two-thirds of the transformants analysed contained multiple tandem integrations of the plasmid. Pulsed-field gel electrophoresis of genomic DNA revealed significant changes in the karyotype of some transformants. Plasmid DNA was frequently detected on more than one chromosome and on mitotically unstable, autonomously replicating elements. Our results show that the introduction of nonhomologous ss DNA into yeast cells can lead to different types of alterations in the yeast genome. Received: 9 February 1996/Accepted: 7 July 1996     

Properties and transforming activities of two plasmids in Streptococcus pneumoniae

Summary Two plasmids from group B streptococcus were introduced into pneumococcus (Streptococcus pneumoniae) and examined for copy number, stability, and some features of the process by which they transform pneumococcal recipients. The 3.6 Mdal pMV158 (tet) was present at a minimum of 12 to 16 copies per chromosome and was never observed to be cured. The 20 Mdal pIP501 (cat erm) had a minimum copy number of 3 to 4 per chromosome and was lost spontaneously at a frequency near 0.03 per division. The presence of novobiocin increased this frequency 2 to 3-fold. Competence for chromosomal transformation and the membrane endonuclease needed for normal DNA entry were required for plasmid transformation. Plasmid transformants segregated transformed cells one generation ahead of chromosomal transformants. Both single and multiple hit components of the transformation reaction kinetics were observed, but the latter could not be seen in the presence of competing chromosomal DNA. The majority of the transforming activity behaved as covalently closed circular DNA in dye-buoyancy gradients. Although most of the activity for both plasmids sedimented in sucrose gradients more rapidly than did monomeric closed circular DNA, a significant fraction was found at a position suggesting that it may have been due to monomeric plasmids.