Natural Genetic Transformation Generates a Population of Merodiploids in Streptococcus pneumoniae

Partial duplication of genetic material is prevalent in eukaryotes and provides potential for evolution of new traits. Prokaryotes, which are generally haploid in nature, can evolve new genes by partial chromosome duplication, known as merodiploidy. Little is known about merodiploid formation during genetic exchange processes, although merodiploids have been serendipitously observed in early studies of bacterial transformation. Natural bacterial transformation involves internalization of exogenous donor DNA and its subsequent integration into the recipient genome by homology. It contributes to the remarkable plasticity of the human pathogen Streptococcus pneumoniae through intra and interspecies genetic exchange. We report that lethal cassette transformation produced merodiploids possessing both intact and cassette-inactivated copies of the essential target gene, bordered by repeats (R) corresponding to incomplete copies of IS861. We show that merodiploidy is transiently stimulated by transformation, and only requires uptake of a ∼3-kb DNA fragment partly repeated in the chromosome. We propose and validate a model for merodiploid formation, providing evidence that tandem-duplication (TD) formation involves unequal crossing-over resulting from alternative pairing and interchromatid integration of R. This unequal crossing-over produces a chromosome dimer, resolution of which generates a chromosome with the TD and an abortive chromosome lacking the duplicated region. We document occurrence of TDs ranging from ∼100 to ∼900 kb in size at various chromosomal locations, including by self-transformation (transformation with recipient chromosomal DNA). We show that self-transformation produces a population containing many different merodiploid cells. Merodiploidy provides opportunities for evolution of new genetic traits via alteration of duplicated genes, unrestricted by functional selective pressure. Transient stimulation of a varied population of merodiploids by transformation, which can be triggered by stresses such as antibiotic treatment in S. pneumoniae, reinforces the plasticity potential of this bacterium and transformable species generally.     

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.     

Genetic Studies of Recombining DNA in Pneumococcal Transformation

The results of genetic fine structure experiments, performed on the amiA locus of Pneumococcus are summarized. The peculiar feature of transformation genetics is that a given donor marker mutation transforms with an efficiency characteristic of the mutated site. In spite of this difficulty, mapping procedures have been devised and quantitative recombination studies performed. It is concluded from these studies that transformation, in this locus, is the consequence of frequent, and essentially random exchanges occurring between donor DNA and the chromosomal DNA of the recipient cell. The average length of uninterrupted donor DNA polynucleotide strand which could be inserted into the chromosome of a transformed cell is estimated, from genetic data, to be probably not greater than 3·10⁵ daltons (for a double-stranded insertion). It is proposed, on the basis of genetic evidence, that following essentially random exchanges between donor DNA and recipient chromosome, a revision process, specific for certain types of mutated sites, occurs. The revision process appears to remove preferentially donor DNA sequences from the primary recombinant structure, and allow repair along the chromosomal template, leading to low efficiency in the genetic integration of these sites. A mechanism for this "destruction-choice" process is presented, and evidence in support of this mechanism discussed.     

Molecular mechanism of genetic recombination in bacterial transformation

Summary B. subtilis cells auxotrophic for two linked markers (ind-his, ind-tyr, his-tyr) have been transformed by means of DNA preparations obtained by hybridization of wild type DNA with the DNA of a strain auxotrophic for one of the linked markers. It was established that hybridization does not increase the transforming activity of DNA for the heterozygous marker. A genetic analysis of the progeny of cells transformed by hybrid or wild type DNA was performed. On the basis of the data obtained a model of genetic recombination in transformation is proved. According to this model both strands of the donor DNA interact independently with the chromosome, and either strand can be incorporated into the cell genome with equal probability. According to the estimate made on the basis of this hypothesis, the probability of integration of a single DNA strand carrying a particular genetic marker is 8%.With 3 Figures in the Text     

Plasmid transformation of Streptococcus sanguis (Challis) occurs by circular and linear molecules

Summary Transformation of Streptococcus sanguis (Challis) by antibiotic resistance plasmids has shown that (a) competente developed with identical kinetics for chromosomal and plasmid DNA; (b) dependence of transformant yield on plasmid DNA concentration was second order; (c) open circular plasmid DNA transformed Challis, although at reduced frequency; (d) linearization of plasmid DNA by restriction enzymes cutting at unique sites inactivated the transforming capacity; (e) transforming activity was restored when linear plasmid molecules generated by different restriction enzymes were mixed; (f) restoration of transforming activity depended on the distance between the linearizing cuts, i.e. on the presence of sufficiently long overlapping homologous sequences; (g) when linear deletion mutants were mixed with linear parental plasmids the smaller plasmid was restored with significantly higher frequency.Based on these data, a model for plasmid transformation of Challis is proposed according to which circular plasmid is linearized during binding and uptake. One DNA strand enters the cell and restoration of circular plasmids inside the cell occurs by annealing of complementary single strands from two different donor molecules. Implications of this model for recombinant DNA experiments in streptococci are discussed.     

In vivo cleavage of transgene donors promotes nuclease‐mediated targeted integration

Targeted DNA integration is commonly used to eliminate position effects on transgene expression. Integration can be targeted to specific sites in the genome via both homology‐based and homology‐independent processes. Both pathways start the integration process with a site‐specific break in the chromosome, typically from a zinc‐finger nuclease (ZFN). We previously described an efficient homology‐independent targeted integration technique that captures short (<100 bp) pieces of DNA at chromosomal breaks created by ZFNs. We show here that inclusion of a nuclease target site on the donor plasmid followed by in vivo nuclease cleavage of both the donor and the chromosome results in efficient integration of large, transgene‐sized DNA molecules into the chromosomal double‐strand break. Successful targeted integration via in vivo donor linearization is demonstrated at five distinct loci in two mammalian cell types, highlighting the generality of the approach. Finally, we show that CHO cells, a cell type recalcitrant to homology‐based integration, are proficient at capture of in vivo‐linearized transgene donors. Moreover, we demonstrate knockout of the hamster FUT8 gene via the simultaneous ZFN‐ or TALE nuclease‐mediated integration of an antibody cassette. Our results enable efficient targeted transgene addition to cells and organisms that fare poorly with traditional homology‐driven approaches. Biotechnol. Bioeng. 2013; 110: 871–880. © 2012 Wiley Periodicals, Inc.     

Evidence for Different Pathways during Horizontal Gene Transfer in Competent Bacillus subtilis Cells

Cytological and genetic evidence suggests that the Bacillus subtilis DNA uptake machinery localizes at a single cell pole and takes up single-stranded (ss) DNA. The integration of homologous donor DNA into the recipient chromosome requires RecA, while plasmid establishment, which is independent of RecA, requires at least RecO and RecU. RecA and RecN colocalize at the polar DNA uptake machinery, from which RecA forms filamentous structures, termed threads, in the presence of chromosomal DNA. We show that the transformation of chromosomal and of plasmid DNA follows distinct pathways. In the absence of DNA, RecU accumulated at a single cell pole in competent cells, dependent on RecA. Upon addition of any kind of DNA, RecA formed highly dynamic thread structures, which rapidly grew and shrank, and RecU dissipated from the pole. RecO visibly accumulated at the cell pole only upon addition of plasmid DNA, and, to a lesser degree, of phage DNA, but not of chromosomal DNA. RecO accumulation was weakly influenced by RecN, but not by RecA. RecO annealed ssDNA complexed with SsbA in vitro, independent of any nucleotide cofactor. The DNA end-joining Ku protein was also found to play a role in viral and plasmid transformation. On the other hand, transfection with SPP1 phage DNA required functions from both chromosomal and plasmid transformation pathways. The findings show that competent bacterial cells possess a dynamic DNA recombination machinery that responds in a differential manner depending if entering DNA shows homology with recipient DNA or has self-annealing potential. Transformation with chromosomal DNA only requires RecA, which forms dynamic filamentous structures that may mediate homology search and DNA strand invasion. Establishment of circular plasmid DNA requires accumulation of RecO at the competence pole, most likely mediating single-strand annealing, and RecU, which possibly down-regulates RecA. Transfection with SPP1 viral DNA follows an intermediate route that contains functions from both chromosomal and plasmid transformation pathways.     

A competence specific inducible protein promotes in vivo recombination in Streptococcus sanguis

Summary We describe the first example of a recombination-specific protein induced during the development of competence for transformation in Streptococcus sanguis. Elaborated in response to stimulation by competence-protein, the 51,000 Molecular Weight (MW) polypeptide is one of at least 10 new polypeptides transiently induced during the competence phase. Biochemical and genetic analyses of the parental, cipA⁺ (competence specific inducible polypeptide A), and mutant, cipA, strains have shown that the 51,000 MW polypeptide has two roles: its low level constitutive synthesis is required for repair of damage to DNA due to UV light and methylmethane sulfonate; its induced synthesis (3–6x10⁴ copies/cell) during the competence phase is essential for promoting recombination between donor single-stranded DNA and the recipient chromosome. Also, ccc plasmid donor DNA transformation, which occurs as a decreasing probability of the increasing donor plasmid MW, requires the inducible function specified by the 51,000 MW polypeptide. The MW independent low level transformation with ccc plasmids, the inheritance of plasmids by conjugation, and the stable maintenance of plasmids introduced by transformation and conjugation, respectively, are independent of the function specified by the 51,000 MW polypeptide.     

Genetic transformation of the Lyme disease agent Borrelia burgdorferi with coumarin-resistant gyrB.

No useful method to genetically manipulate Borrelia burgdorferi, the causative agent of Lyme disease, has been developed previously. We have used resistance to the coumarin antibiotic coumermycin A1, an inhibitor of DNA gyrase, as a genetic marker to monitor the transformation of B. burgdorferi by electroporation. Introduction of site-directed mutations into the gyrB gene demonstrated that transformation was successful, provided evidence that homologous recombination occurs on the chromosome, and established that mutations at Arg-133 of DNA gyrase B confer coumermycin A1 resistance in B. burgdorferi. The coumermycin A1-resistant gyrB marker and genetic transformation can now be applied toward dissecting the physiology and pathogenesis of the Lyme disease agent on a molecular genetic level.     

The synergistic effects of SV40 and BUdR on induction of gene mutations and chromosomal aberrations in chinese hamster cells

The induction of chromosomal abberations and gene mutations was studied in Chinese hamster cells after separate and combined treatment with BUdR and SV40. Separate treatment of cells with BUdR or virus infection increased the yield of chromosomal aberrations and reversions from glutamine requirement, expressed at 40°C (a ts mutant), to prototrophy. The combined effect of the incorporation of BUdR into one DNA strand, and a subsequent infection by SV40 was additive as regards the percentage of aberrant metaphases. The integration of the analogue into both DNA strands followed by SV40 treatment resulted in a statistically significant increase in the frequency of aberration-carrying metaphases, as compared with the frequency expected if the two agents had acted additively. The same phenomenon was detected when the frequency of reversions to glutamine independence was studied. Hence, the effect of the joint treatment by BUdR incorporated into both DNA strands and SV40 was synergistic. This is known to characterize the effect of BUdR on virus-induced transformation. Therefore, obviously the agent that enhances the malignant transformation of cells by the virus similarly modifies its mutagenic activity.

The results obtained are presumed to confirm the previously advanced hypothesis that the same events following infection might control both the integration of viral DNA into the host-cell chromosome (and hence cell transformation) and virus-induced mutagenesis. The role of repair processes in the synergistic effect of BUdR and SV40 in the yield of reversions to glutamine independence is discussed.     

Non-reciprocal chromosomal bridge-induced translocation (BIT) by targeted DNA integration in yeast

Several experimental in vivo systems exist that generate reciprocal translocations between engineered chromosomal loci of yeast or Drosophila, but not without previous genome modifications. Here we report the successful induction of chromosome translocations in unmodified yeast cells via targeted DNA integration of the KAN^R selectable marker flanked by sequences homologous to two chromosomal loci randomly chosen on the genome. Using this bridge-induced translocation system, 2% of the integrants showed targeted translocations between chromosomes V-VIII and VIII-XV in two wild-type Saccharomyces cerevisiae strains. All the translocation events studied were found to be non-reciprocal and the fate of their chromosomal fragments that were not included in the translocated chromosome was followed. The recovery of discrete-sized fragments suggested multiple pathway repair of their free DNA ends. We propose that centromere-distal chromosome fragments may be processed by a break-induced replication mechanism ensuing in partial trisomy. The experimental feasibility of inducing chromosomal translocations between any two desired genetic loci in a eukaryotic model system will be instrumental in elucidating the molecular mechanism underlying genome rearrangements generated by DNA integration and the gross chromosomal rearrangements characteristic of many types of cancer.Electronic Supplementary Material Supplementary material is available for this article at     

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.     

Initiation of DNA replication in Bacillus subtilis. V. Role of DNA gyrase and superhelical structure in initiation

Summary When spores of a thymine-requiring mutant of Bacillus subtilis were germinated in a medium lacking thymine, an initiation potential (an ability to initiate and complete one round of replication in the presence of thymine and in the absence of protein and RNA synthesis) was formed for both chromosomal and plasmid replication. The effect of two inhibitors of DNA gyrase, novobiocin (Nov) and nalidixic acid (Nal), on the initiation potential formed during germination for chromosomal and plasmid replication was examined.Nov and Nal inhibited formation of the initiation potential completely if the drug was added at the onset of germination. In contrast, initiation of chromosomal and plasmid replication occurred in the presence of DNA gyrase inhibitors when the drug was added after the initiation potential had been fully formed. However, chromosomal replication initiated in the presence of the inhibitors ceased after a fragment of approximately 15 MD (15×10⁶ daltons) had been replicated, and plasmid replication was limited to one round of replication in approximately half of the plasmid molecules present in the spores.Furthermore the initiation potential for both chromosomal and plasmid replication though established was destroyed gradually but steadily by prolonged incubation with Nov in the absence of thymine. In addition, relaxation of the superhelical structure of plasmid DNA during incubation with Nov was observed in vivo. This relaxation was blocked by ethidium bromide, which dissociated the S-complex. On the other hand, incubation with Nal did not reduce the initiation potential nor did it change the superhelicity of the plasmid DNA in vivo. This is consistent with the known effect of gyrase inhibitors on the enzymatic activity of DNA gyrase.These results clearly demonstrate that both the action of DNA gyrase and the superhelical structure of the DNA are essential for the initiation of chromosomal and plasmid replication. The specific chromosome organization essential for initiation and elongation and the role of DNA gyrase are discussed.IV of this series is Yoshikawa et al. 1980     

Genetic and molecular events in transformation ofHaemophilus influenzae with plasmid RSF 0885 carrying cloned segments of chromosomal DNA

InHaemophilus influenzae genetic transformation for a plasmid marker is significantly increased when recombinant plasmid RSF 0885 DNA carrying chromosomal DNA segments is used instead of the plasmid DNA alone. Chromosomal DNA by itself, added even a few minutes after the addition of plasmid DNA to competent cells, stopped further uptake of the plasmid DNA. These observations are consistent with the idea that plasmid RSF 0885 contains a ‘degenerate’ version of the required eleven base-pair ‘uptake sequence’ inHaemophilus. The transformation activity of the recombinant plasmid DNA is recoverable after its entry into cells, although the specific biological activity of the re-isolated plasmid DNA is less than that of the parental recombinant plasmid DNA. Therec 1 gene function of the host is necessary for obtaining higher transformation frequencies with recombinant DNA from five different clones. The reduced transformation frequencies seen inrec 1 ^- strain is not all due to a permanent damage to the donor DNA since the recovered recombinant plasmid DNA from such cells can increase the transformation efficiency onrec 1 ⁺ strain.     

Mechanism of transformation in B. subtilis

Summary Transformation in B. subtilis is achieved by the uptake of donor DNA into recipient cells and the integration of part of this donor DNA into the host chromosome. The evidence presented in this report is interpreted to indicate that donor double helical DNA, on entry into host cells is rapidly membrane bound and can remain in this state for a consicerable time, perhaps even until integration. This bound DNA consists of molecules which have been reduced in size and degraded on uptake, and appear as partially single-stranded molecules. It is suggested that the donor DNA initially forms single strands which rapidly assume a partially single stranded nature by association with the host DNA or by reannealing.Host cells, by virtue of the competent state, possess temporarily, and prior to the addition of donor DNA, chromosomes with single-stranded gaps. It is likely that such gaps are larger than the single-stranded segments of donor DNA which are to be integrated. Results are described which are best explained if integration is achieved by an initial annealing between the single-stranded donor and host segments followed by their covalent linkage.     

Natural transformation of Neisseria gonorrhoeae: from DNA donation to homologous recombination

Gonococci undergo frequent and efficient natural transformation. Transformation occurs so often that the population structure is panmictic, with only one long-lived clone having been identified. This high degree of genetic exchange is likely necessary to generate antigenic diversity and allow the persistence of gonococcal infection within the human population. In addition to spreading different alleles of genes for surface markers and allowing avoidance of the immune response, transformation facilitates the spread of antibiotic resistance markers, a continuing problem for treatment of gonococcal infections. Transforming DNA is donated by neighbouring gonococci by two different mechanisms: autolysis or type IV secretion. All types of DNA are bound non-specifically to the cell surface. However, for DNA uptake, Neisseria gonorrhoeae recognizes only DNA containing a 10-base sequence (GCCGTCTGAA) present frequently in the chromosome of neisserial species. Type IV pilus components and several pilus-associated proteins are necessary for gonococcal DNA uptake. Incoming DNA is subject to restriction, making establishment of replicating plasmids difficult but not greatly affecting chromosomal transformation. Processing and integration of transforming DNA into the chromosome involves enzymes required for homologous recombination. Recent research on DNA donation mechanisms and extensive work on type IV pilus biogenesis and recombination proteins have greatly improved our understanding of natural transformation in N. gonorrhoeae. The completion of the gonococcal genome sequence has facilitated the identification of additional transformation genes and provides insight into previous investigations of gonococcal transformation. Here we review these recent developments and address the implications of natural transformation in the evolution and pathogenesis N. gonorrhoeae.     

Transformation ofHaemophilus influenzae following electroporation with plasmid and chromosomal DNA

Naturally transformable species, such asHaemophilus influenzae, have evolved systems for the efficient uptake and integration of DNA from the surrounding environment. We compared this competence-dependent DNA uptake system to electroporation, which has been widely used in the past few years to introduce DNA into cells, for transformingHaemophilus influenzae. Electroporation improved transformation efficiency when noncompetent cells were used and when DNA lackingHaemophilus-specific uptake sequences was used for transformation of competent cells. An increase in plasmid-to-chromosome recombination was seen when plasmid DNA containing chromosomal inserts was introduced into competent cells by electroporation, as observed previously for natural transformation.