Excision and repair of mismatched base pairs in transformation of Streptococcus pneumoniae

Summary The use of heteroduplex DNA molecules as donors in pneumococcal transformation makes it possible to follow the fate of each DNA strand. The integration efficiency of each strand depends strongly upon the single base changes it carries. The function (hex) which reduces drastically the transformation yield of markers referred to as low efficiency (LE) tends to remove either donor strand without respect to which one is introduced. In the case of high efficiency (HE) markers the reduction in the transformation yield involves the elimination of only one donor strand. For a given locus it can be either one depending upon the mutation. The reduction in transformation yield can be less drastic for HE markers than for both strands of the LE markers. These data are discussed in terms of differences in the affinity for mismatched base pairs.We have studied the transfer of information from each donor DNA strand to the recipient genome, on the basis of differences in the rates of phenotypic expression of a given marker introduced on opposite strands. Results show that, as in the case of LE markers, the information from HE markers, when introduced on the strand recognized by the hex function, is transmitted to both strands of the recipient molecule. Correction of the recipient strand to homozygosis probably accounts for this information transfer. These results, together with earlier investigations, strongly suggest that the hex function is an excision-repair system acting on donor-recipient base pair mismatches.     

Destruction of low efficiency markers is a slow process occurring at a heteroduplex stage of transformation

Summary Direct evidence is presented that the mechanism which discriminates against low efficiency markers in transformation of Diplococcus pneumoniae of genotype hex ⁺ acts on them after the formation of donor-recipient heteroduplexes. This conclusion is based on assays of the transforming activity of donor markers in lysates made after various times of incubation of recipient cells following exposure to DNA. The activity of a low efficiency marker rises substantially, indicating formation of native-like heteroduplex structures, and then falls. At 37° C the process is essentially completed 10 minutes after entry, and the apparent half life of a susceptible heteroduplex is 1.5 to 2 minutes. Data from these and other experiments imply that about as many of the surviving low efficiency markers have simply escaped attack as have been inserted into both strands by the excision-repair process suggested by Ephrussi-Taylor.     

The nature of the transformation process in Escherichia coli K12

Summary The nature of the transformation process in E. coli was studied by investigating various factors which affect the efficiency of transformation. CaCl₂ treatment of the recipient cells is absolutely necessary for transformation and the optimum concentration was found to be 30 mM. The efficiency of transformation is dependent upon temperature during incubation of the recipient cells with DNA. The efficiency is also affected by the molecular weight of donor DNA used. Sheared DNA with molecular weights ranging from 10 to 30x10⁶ daltons was most efficient, increasing the number of transformants by a factor of 5 to 10 as compared to unsheared DNA. The intracellular status of recB-recC DNase (ATP-dependent DNase) is another important factor which determines the transformability of E. coli K12. This was shown by demonstrating that a recB ^- recC ^- sbcA ^- strain was transformable as well as the previously demonstrated recB ^- recC ^- sbcB ^- strain. Therefore, it seems reasonable to conclude that the E. coli K12 strain is transformable if the ATP-dependent DNase is absent or diminished in function and a state of recombinational proficiency exists.     

The mechanism of competence in the transformation of streptococci of serological group H

Summary The production of a special competence factor (cpf) by the recipient cells is one of the key-elements of the complex mechanims of competence in transformation. The role of cpf and its interrelation with the other factors involved in competence remain obscure. The evidence regarding the genetic background of competence is also very scarce.The cpf production ability was demonstrated to be a genetic unit which could be transferred in transformation. The cpf marker was transformed in a heterospecific reaction, in which the S. challis (cpf ⁺) strain was the DNA donor, and S. wicky (cpf ^–) its recipient.As a result of the incorporation of this marker, S. wicky cells acquired the stable cpf production ability and, consequently, the transformation ability in a yield equal to that shown by the donor cells. DNA isolated from the S. wicky (cpf ⁺) transformants could be applied, in turn, as a donor of the cpf marker. The experiments were performed by a semiquantitative technique and the yield of the transfer of the cpf marker amounted to about 1%.     

Homology in capsular transformation reactions in Pneumococcus

Summary Experiments were carried out to determine the relative effect of homology inside or outside of the capsular genomes of donor and recipient strains of pneumococci on the frequency of transfer of capsular markers. In one series of experiments, 3 recipient strains were transformed to CapIII⁺ by DNA from 2 donor strains. Recipient strains (III)capIII D6 ¹, (II)capIII D15 P1 ¹, and (II)capII-1 ¹ were each transformed to CapIII⁺ at different absolute frequencies dependent upon the amount of genetic information that the strain had to acquire. The chromosomal background of the donor strain carrying the CapIII capsular genome had no influence on the results, however, for each strain was transformed at the same frequency by DNA from donor strain (II)CapIII⁺ or donor strain (III)CapIII⁺. In a second series of experiments, 2 (I)CapIII-strains, a (II)CapIII-strain and a (III)CapIII-strain were transformed to heterologous type I and binary type I-III with DNA from donor strains (I)CapI⁺, (II)CapI⁺, and (III)CapI⁺. Again, the chromosomal background of the donor strain was unimportant to the results. The origin of the recipient strain, however, markedly influenced the frequency of transformation. (I)CapIII-strains were transformed to CapI⁺ at about 10 times the frequency and to CapI-III at from 18–6000 times the frequency of the other CapIII-strains. Consideration of the results leads to the conclusion that transformation of CapIII-strains to CapI⁺ and transformation of CapI-strains to CapIII⁺ are not reciprocal reactions; CapI-strains lose less information in transformation to CapIII⁺ than CapIII-strains gain in transformation to CapI⁺. In (I)CapIII-recipient strains, the residual information from the CapI capsular genome is responsible for the higher frequency of transformation to both CapI⁺ and to CapI-III. It is suggested that addition of exogenous linear DNA to a recipient chromosome to give rise to binary strains occurs when sequence homology with the recipient is limited to one end of a piece of transforming DNA. Models to explain the results (Figs. 1 through 3) are consistent with the experimental findings and are amenable to further testing.     

Identification of Streptococcus pneumoniae mismatch repair genes by an additive transformation approach

Summary During transformation of Streptococcus pneumoniae, mismatch repair occurs on donor-recipient heteroduplexes harboring some mismatched base pairs. A few mutants defective in mismatch repair have been isolated and termed hex ^-. However, neither the number of genes involved nor their products have yet been identified. In an attempt to characterize such genes we have used an additive transformation approach — that is the inactivation of genes by insertion of chimeric plasmids. Pneumococcal DNA fragments were joined in vitro to a plasmid derivative of pBR325 that carries an erythromycin resistance determinant and does not replicate autonomously in S. pneumoniae. Ery-r transformants obtained with such a ligation mixture arise via homology-dependent integration of the chimeric plasmids into the chromosome. Hex ^- mutants have been selected among the ery-r population. Comparison of these mutants by Southern blot hybridization with a vector probe reveals that at least two genes are involved in mismatch repair.     

A Fast and Practical Yeast Transformation Method Mediated by Escherichia coli Based on a Trans-Kingdom Conjugal Transfer System: Just Mix Two Cultures and Wait One Hour

Trans-kingdom conjugation is a phenomenon by which DNA is transferred into a eukaryotic cell by a bacterial conjugal transfer system. Improvement in this method to facilitate the rapid co-cultivation of donor bacterial and recipient eukaryotic cell cultures could make it the simplest transformation method, requiring neither isolation of vector DNA nor preparation of competent recipient cells. To evaluate this potential advantage of trans-kingdom conjugation, we examined this simple transformation method using vector combinations, helper plasmids, and recipient Saccharomyces cerevisiae strains. Mixing donor Escherichia coli and recipient S. cerevisiae overnight cultures (50 μL each) consistently yielded on the order of 10¹ transformants using the popular experimental strain BY4742 derived from S288c and a shuttle vector for trans-kingdom conjugation. Transformation efficiency increased to the order of 10² using a high receptivity trans-kingdom conjugation strain. In addition, either increasing the amount of donor cells or pretreating the recipient cells with thiols such as dithiothreitol improved the transformation efficiency by one order of magnitude. This simple trans-kingdom conjugation-mediated transformation method could be used as a practical yeast transformation method upon enrichment of available vectors and donor E. coli strains.     

Fate of transforming DNA following uptake by competent Bacillus subtilis

Summary During transformation in Bacillus subtilis, donor and recipient DNA are initially associated by non-covalent bonds. The donor and recipient moieties later become covalently joined. The molecular weight of the donor component, when freed from the noncovalent complex by sucrose gradient sedimentation under alkaline conditions, ranges from 1 to 5×10⁶, with an average of about 2.5 to 3.0×10⁶. The latter values are in good agreement with previous measurements of the size of the integrated donor fragment.     

Fate of Donor Deoxyribonucleic Acid in a Highly Transformation-Deficient Strain of Haemophilus influenzae

A transformation-deficient strain of Haemophilus influenzae (efficiency of transformation 10⁴-fold less than that of the wild type), designated TD24, was isolated by selection for sensitivity to mitomycin C. In its properties the mutant was equivalent to recA type mutants of Escherichia coli. The TD24 mutation was linked with the str-r marker (about 30%) and only weakly linked with the nov-r2.5 marker. The uptake of donor deoxyribonucleic acid (DNA) was normal in the TD24 strain, but no molecules with recombinant-type activity (molecules carrying both the donor and the resident marker) were formed. In the mutant the intracellular presynaptic fate of the donor DNA was the same as that in the transformation-proficient (wild-type) strain, and the radioactive label of the donor DNA associated covalently with the recipient chromosome in about the same quantity as in the wild type. However, many fewer donor atoms were associated with segments of the mutant's recipient chromosome as compared with segments of the wild-type chromosome. In the mutant the association was accompanied by complete loss of donor marker activity. The lack of donor marker activity of the donor-recipient complex of DNA isolated from the mutant was not due to lack of uptake of the complex by the second recipient and its inability to associate with the second recipient's chromosome. Because the number of donor-atom-carrying resident molecules was higher than could be accounted for by the lengths of presynaptic donor molecules, we favor the idea that the association of donor DNA atoms with the mutant chromosome results from local DNA synthesis rather than from dispersive integration of donor DNA by recombination.     

DNA repair and the evolution of transformation IV. DNA damage increases transformation

Natural genetic transformation in the bacterium Bacillus subtilis provides a model system to explore the evolutionary function of sexual recombination. In the present work, we study the response of transformation to UV irradiation using donor DNAs that differ in sequence homology to the recipient's chromosome and in the mechanism of transformation. The four donor DNAs used include homologous-chromosomal-DNA, two plasmids containing a fragment of B. subtilis trp⁺ operon DNA and a plasmid with no sequence homology to the recipient cell's DNA. Transformation frequencies for these DNA molecules increase with increasing levels of DNA damage (UV radiation) to recipient cells, only if their transformation requires homologous recombination (i.e. is recA⁺-dependent). Transformation with non-homologous DNA is independent of the recipient's recombination system and transformation frequencies for it do not respond to increases in UV radiation. The transformation frequency for a selectable marker increases in response to DNA damage more dramatically when the locus is present on small, plasmid-borne, homologous fragments than if it is carried on high molecular weight chromosomal fragments. We also study the kinetics of transformation for the different donor DNAs. Different kinetics are observed for homologous transformation depending on whether the homologous locus is carried on a plasmid or on chromosomal fragments. Chromosomal DNA- and non-homologous-plasmid-DNA-mediated transformation is complete (maximal) within several minutes, while transformation with a plasmid containing homologous DNA is still occurring after an hour. The results indicate that DNA damage directly increases rates of homologous recombination and transformation in B. subtilis. The relevance of these results and recent results of other labs to the evolution of transformation are discussed.     

An unstable donor-recipient DNA complex in transformation of Bacillus subtilis.

Summary In re-extracted DNA obtained shortly after uptake of transforming DNA by Bacillus subtilis, increased amounts of donor DNA radioactivity banding at the position of donor-recipient DNA complex (DRC) are observed in CsCl gradients, if the cells are irradiated with high doses of UV prior to reextraction of the DNA. Qualitatively, the same phenomenon is observed if lysates of transforming cells are irradiated. UV-irradiation of lysates of competent cells to which single-stranded DNA is added after lysis, does not result in linkage of this DNA to the chromosomal DNA. Two observations argue in favour of the formation of a specific labile complex between donor and resident DNA during transformation. Firstly, heterologous donor DNA from Escherichia coli, although being processed to single-stranded DNA in competent B. subtilis, does not seem to be linked to the recipient chromosome upon UV-irradiation, and secondly, the labile complex of donor and recipient DNA can be stabilized by means of treatment of the lysates of transforming cells with 4, 5¹, 8-trimethylpsoralen in conjuction with long-wave ultra violet light irradiation. This indicates that base-pairing is involved in the formation of the complex. On the basis of these results we assume that the unstable complex of donor and recipient DNA is an early intermediate in genetic recombination during transformation.     

Restriction of plasmid-mediated transformation in Bacillus subtilis 168.

Summary When plasmids carrying leucine genes of Bacillus subtilis 168 were isolated from a restriction and modification deficient (r^-m^-) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec ⁺ strain (transformation by integration of the donor DNA into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r⁺ or r^- phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r^-m^- strains should be used for construction of recombinant DNA molecules in B. subtilis 168.     

Conditions for quantitative transformation inAcinetobacter calcoaceticus

A transformation procedure that yielded high efficiencies was developed forAcinetobacter calcoaceticus. Strain BD413 Ura⁻ trpE was transformed to tryptophan prototrophy using highly purified DNA. Experimental parameters studied were: (i) recipient cell concentration, (ii) DNA concentration, (iii) growth phase of the recipient cell population, (iv) composition of the growth and transforming medium, and (v) time of incubation of recipient cells with donor DNA. Strain BD413 was competent for transformation throught the growth cycle, with highest competence occurring early in the exponential phase of growth. Maximal transformation efficiencies of 0.5% to 0.7% were obtained in media supporting rapid growth. Recipient cell concentrations of 1×10⁶ to 6×10⁶ cells/ml yielded the highest transformation frequencies, regardless of DNA concentration.     

A colony‐to‐lawn method for efficient transformation of Escherichia coli

Aims: To develop a fast, convenient, inexpensive and efficient Escherichia coli transformation method for changing hosts of plasmids, which can also facilitate the selection of positive clones after DNA ligation and transformation. Methods and Results: A single fresh colony from plasmid‐containing donor strain is picked up and suspended in 75% ethanol. Cells are pelleted and resuspended in CaCl₂ solution and lysed by repetitive freeze–thaw cycles to obtain plasmid‐containing cell lysate. The E. coli recipient cells are scraped from the lawn of LB plate and directly suspended in the plasmid‐containing cell lysate for transformation. Additionally, a process based on colony‐to‐lawn transformation and protein expression was designed and conveniently used to screen positive clones after DNA ligation and transformation. Conclusions: With this method, a single colony from plasmid‐containing donor strain can be directly used to transform recipient cells scraped from lawn of LB plate. Additionally, in combination with this method, screening of positive clones after DNA ligation and transformation can be convenient and time‐saving. Significance and Impact of the Study: Compared with current methods, this procedure saves the steps of plasmid extraction and competent cell preparation. Therefore, the method should be highly valuable especially for high‐throughput changing hosts of plasmids during mutant library creation.     

On the Nature of Recombinants Formed during Transformation in Hemophilus influenzae

During the process of transformation in Hemophilus influenzae integration of donor DNA, i.e. the formation of recombinant DNA, involves the incorporation of single-stranded DNA. Evidence was obtained from cesium chloride density gradient centrifugation of DNA from donor-recipient complexes that integration was accompanied by the formation of hybrid DNA with a density intermediate with respect to heavy, ²H, ¹⁵N, donor and light, ¹H, ⁴N recipient DNA. On denaturation the position of the heavy donor DNA moved closer to, but not all the way toward, the density position of the original donor DNA. In addition to supporting the idea of single-stranded incorporation, this evidence suggested that the integrated donor DNA was covalently linked to light recipient DNA. The DNA was taken up in the double-stranded form and no detectable amounts of denatured DNA could be found during the transformation process. However, during the process of integration an amount of donor atoms, equivalent to the amount of hybrid DNA formed, appeared in recipient DNA, and indicated that while one strand of DNA was integrated the other was broken down and resynthesized. The density of the hybrid DNA, as well as rebanding of denatured hybrid, indicated that the size of the integrated piece of DNA was large, approximately 6 x 10⁶ daltons.     

Use of a cloned DNA fragment to analyze the fate of donor DNA in transformation of Streptococcus pneumoniae

The integration of donor label into the recipient fragment is followed during transformation of Streptococcus pneumoniae. The method used involves gel analysis of restriction endonuclease-treated recipient DNA after recombination with a radioactively labeled homologous cloned fragment.     

Exchange of chromosomal markers by natural transformation between the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell

Both the soil isolate,Pseudomonas stutzeri JM300, and the marine isolate,Pseudomonas stutzeri strain ZoBell, have been shown previously to be naturally transformable. This study reports the detection of genetic exchange by natural transformation between these two isolates. Transformation frequency was determined by filter transformation procedures. Three independent antibiotic resistance loci were used as chromosomal markers to monitor this exchange event: resistance to rifampicin, streptomycin, and nalidixic acid. The maximum frequencies of transformation were on the order of 3.1 to 3.8×10^-6 transformants per recipient; frequencies over an order of magnitude greater than those for spontaneous antibiotic resistance, although they are lower than those observed for soil: soil or marine: marine strain crosses. This exchange was inhibited by DNase I. Transformation was observed between soil and marine strains, both by filter transformation using purified DNA solutions and when transforming DNA was added in the form of viable donor cells. The results from this study support the close genetic relationship betweenP. stutzeri JM300 andP. stutzeri strain ZoBell. These results also further validate the utility ofP. stutzeri as a benchmark organism for modeling gene transfer by natural transformation in both soil and marine habitats.     

Direct visualization of Agrobacterium‐delivered VirE2 in recipient cells

Agrobacterium tumefaciens is a natural genetic engineer widely used to deliver DNA into various recipients, including plant, yeast and fungal cells. The bacterium can transfer single‐stranded DNA molecules (T–DNAs) and bacterial virulence proteins, including VirE2. However, neither the DNA nor the protein molecules have ever been directly visualized after the delivery. In this report, we adopted a split‐GFP approach: the small GFP fragment (GFP11) was inserted into VirE2 at a permissive site to create the VirE2‐GFP11 fusion, which was expressed in A. tumefaciens; and the large fragment (GFP1–10) was expressed in recipient cells. Upon delivery of VirE2‐GFP11 into the recipient cells, GFP fluorescence signals were visualized. VirE2‐GFP11 was functional like VirE2; the GFP fusion movement could indicate the trafficking of Agrobacterium‐delivered VirE2. As the natural host, all plant cells seen under a microscope received the VirE2 protein in a leaf‐infiltration assay; most of VirE2 moved at a speed of 1.3–3.1 μm sec^?1 in a nearly linear direction, suggesting an active trafficking process. Inside plant cells, VirE2‐GFP formed filamentous structures of different lengths, even in the absence of T‐DNA. As a non‐natural host recipient, 51% of yeast cells received VirE2, which did not move inside yeast. All plant cells seen under a microscope transiently expressed the Agrobacterium‐delivered transgene, but only 0.2% yeast cells expressed the transgene. This indicates that Agrobacterium is a more efficient vector for protein delivery than T‐DNA transformation for a non‐natural host recipient: VirE2 trafficking is a limiting factor for the genetic transformation of a non‐natural host recipient. The split‐GFP approach could enable the real‐time visualization of VirE2 trafficking inside recipient cells.     

Genetic defects in DNA repair system and enhancement of intergenote transformation efficiency in Bacillus subtilis Marburg.

Summary Mechanisms of inefficiency in heterospecies transformation were studied with a transformation system consisting of Bacillus subtilis 168TI (trpC2 thy) as recipient and of DNA prepared from partially hybrid strains of B. subtilis which had incorporated trp ⁺ DNA of B.amyloliquefaciens 203 (formerly, B.megaterium 203) in the chromosome (termed intergenote). The intergenote transformation was not so efficient as the corresponding homospecies transformation and the efficiency appeared to relate inversely with the length of heterologous portion in the intergenote. When a variety of ultraviolet light (UV) sensitive mutants, deficient in host-cell reactivation capacity, were used as recipients for the intergenote transformation, 2 out of 16 mutants exhibited significantly enhanced transformation efficiency of the trpC marker. Genetic studies by transformation showed that the trait relating to the enhancement of intergenote-transformation efficiency was always associated with the UV sensitivity, suggesting that these two traits are determined by a single gene. The efficiency of intergenote transformation was highly affected also by DNAconcentration; the lower the concentration, the less the efficiency. When, however, the UV sensitive mutant was used as recipient, the effect of DNA concentration was largely diminished, suggesting the reduction of DNA-inactivating activity in the UV sensitive recipient. These results were discussed in relation to a possible excision-repair system selectively correcting the mismatched DNA in in the course of intergenote transformation.This work was supported by a Grant-in-Aid for scientific research from the Ministry of Education, Japan.