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PubMed1998

Formation of single-stranded DNA during DNA transformation of Neisseria gonorrhoeae.

Neisseria gonorrhoeae is naturally competent for DNA transformation. In contrast to other natural prokaryotic DNA transformation systems, single-stranded donor DNA (ssDNA) has not previously been detected during transformation of N. gonorrhoeae. We have reassessed the physical nature of gonococcal transforming DNA by using a sensitive nondenaturing native blotting technique that detects ssDNA. Consistent with previous analyses, we found that the majority of donor DNA remained in the double-stranded form, and only plasmid DNAs that carried the genus-specific DNA uptake sequence were sequestered in a DNase I-resistant state. However, when the DNA was examined under native conditions, S1 nuclease-sensitive ssDNA was identified in all strains tested except for those bacteria that carried the dud-1 mutation. Surprisingly, ssDNA was also found during transformation of N. gonorrhoeae comA mutants, which suggested that ssDNA was initially formed within the periplasm.

Bacterial Proteins↗
PubMed1986

Heterologous insertion of transforming DNA and generation of new deletions associated with transformation in Aspergillus nidulans.

The analysis of four transformants for the proline catabolism (prn) gene cluster of Aspergillus nidulans is reported. Using a combination of traditional genetic methodology and Southern hybridisation we have shown that in two cases multiple copies of the transforming plasmid have been integrated into linkage groups other than VII, which contains the prn cluster. In the other two cases integration of the plasmid has probably occurred homologously. The phenotype of these transformants is broadly consistent with increased copy number resulting in increased expression. Genetic manipulation of these transformants using the sexual or parasexual cycles has shown that recombination events during and possibly also subsequent to integration of the transforming DNA can generate new mutational lesions, in particular, deletions.

Aspergillus nidulans↗
PubMed1988

Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation.

The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.

Amidohydrolases↗
PubMed1991

Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast.

The methods described in this chapter permit the manipulation of virtually any cloned yeast chromosomal sequence by virtue of the fact that DNA transformed into yeast integrates into the chromosome by homologous recombination. Furthermore, double-strand breaks in transforming DNA stimulate recombination and can be used to target integration events. This allows simple one-step gene disruption methods using yeast selectable markers. The availability of counterselectable markers makes it possible to replace chromosomal sequences with mutant alleles that cannot be directly selected. Finally, these same methods can be used to rescue chromosomal alleles on plasmids for subsequent molecular analysis.

Alleles↗
PubMed1999

Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili.

We have recently described the expression of two pili of different lengths on the surface of Legionella pneumophila (B. J. Stone and Y. Abu Kwaik, Infect. Immun. 66:1768-1775, 1998). Production of long pili requires a functional pilEL locus, encoding a type IV pilin protein. Since type IV pili in Neisseria gonorrhoeae are associated with competence for DNA transformation, we examined the competence of L. pneumophila for DNA transformation under conditions that allowed the expression of type IV pili. We show that L. pneumophila is naturally competent for DNA transformation by isogenic chromosomal DNA and by plasmid DNA containing L. pneumophila DNA. Many different L. pneumophila loci are able to transform L. pneumophila after addition of plasmid DNA, including gspA, ppa, asd, and pilEL. The transformation frequency is reduced when competing DNA containing either L. pneumophila DNA or vector sequences is added to the bacteria, suggesting that uptake-specific sequences may not be involved in DNA uptake. Competence for DNA transformation correlates with expression of the type IV pili, and a pilEL mutant defective in expression of type IV pili is not competent for DNA transformation. Complementation of the mutant for competence is restored by the reintroduction of a cosmid that restores production of type IV pili. Minimal competence is restored to the mutant by introduction of pilEL alone. We conclude that competence for DNA transformation in L. pneumophila is associated with expression of the type IV pilus and results in recombination of L. pneumophila DNA into the chromosome. Since expression of type IV pili also facilitates attachment of L. pneumophila to mammalian cells and protozoa, we designated the type IV pili CAP (for competence- and adherence-associated pili).

Bacterial Proteins↗
PubMed1978

Fate of heterospecific transforming DNA bound to Streptococcus sanguis.

The fate of 3H-labeled str-r fus-s DNA from Streptococcus pneumoniae, bound after a 1-min uptake to 14C-labeled str-s fus-r S. sanguis recipients, was followed by techniques previously developed for analyzing the fate of homospecific DNA. Heterospecific S. pneumoniae DNA was bound and formed complexes with recipient protein in a manner similar to that of homospecific DNA but transformed relatively poorly. The rate at which complexed heterospecific DNA becomes physically associated with recipient DNA, and at which donor markers are integrated into the chromosome, was slower than in the case of homospecific DNA. In addition, about half of the heterospecific donor counts initially bound in trichloracetic acid-insoluble form were gradually solubilized and released from the cell. The association of heterospecific DNA with the recipient chromosome was more unstable than that involving homospecific DNA, since only associations of the former type were largely dissociated by isolation and resedimentation. The donor DNA-containing material so dissociated had the same sedimentation properties as complexed heterospecific DNA before association, indicating that the complex of single-stranded donor DNA and recipient protein formed on uptake moves as a whole from its site of formation to synapse with the chromosome.

Bacterial Proteins↗
PubMed1979

Effects of varying O2 concentration on the X-ray sensitivity of transforming DNA.

The X-ray-induced inactivation of the biological activity of Bacillus subtilis transforming DNA in dilute aqueous solution has been studied over a wide range of O2 concentrations in an attempt to elucidate the mechanisms involved in O2 action. When the DNA is irradiated in the presence of 100 per cent O2 there is a protection of the transforming DNA compared to the sensitivity in N2-saturated or in N2O-saturated solutions. When the equilibrating gas contains intermediate concentrations of O2 (1 per cent--90 per cent) in N2 or N2O, the DNA sensitivity is equivalent to that in pure N2 or N2O respectively. At low O2 concentrations (approximately 0.14 per cent O2 in N2 or in N2O) there is a sensitization of the DNA and this sensitization can be prevented by .OH scavengers. Possible mechanisms for these actions of O2 on the radiation sensitivity of transforming DNA are discussed.

Bacillus subtilis↗
PubMed1989

Osmolability of Escherichia coli and modification of [125I]ampicillin-binding by competence induction for uptake of transforming DNA.

The regimen conferring competence for uptake of transforming DNA is shown to render Escherichia coli osmolabile. Three different K-12 strains were exposed to the standard procedure of competence induction, i.e. incubation in the presence of 0.1 M Ca2+ or Mg2+ for 50 min at 0 degrees C, interrupted by a heat shock for 5 min at 37 degrees C. Upon osmotic challenge of competent cells formation of protoplasts was observed in approximately 2% of the treated cells. Incubation of competent cells of strain W1485 in phosphate-buffered saline for 1, 2, and 3 h reduced the viable counts to 67, 58, and 41%, respectively. Competence induction with divalent cations altered the affinity of penicillin-binding proteins (PBPs) for [125I]ampicillin. In isolated cell envelopes the presence of Ca2+ and Mg2+ stimulated the binding of [125I]ampicillin to PBPs 1, 3, 4, 5, and 6, whereas the binding to PBP 2 remained unchanged. The binding to PBP 1 C was inhibited by 0.23 M Ca2+. In living cells the binding to PBPs 1, 3, and 4 was enhanced, while the binding to PBP 8 was inhibited. Newly [125I]ampicillin-labelled proteins of Mr 55,000 and 45,000 were apparent, especially after competence induction with Ca2+. Interaction of divalent cations with PBPs is suggested to contribute to osmolability of competent cells. Disintegration of the cell wall may be necessary for uptake of transforming DNA.

Ampicillin↗
PubMed1981

Effects of oxygen and sulphydryl-containing compounds on irradiated transforming DNA. Part I. Actions of dithiothreitol.

The actions and interactions of oxygen and the sulphydryl-containing compound dithiothreitol (DTT) upon the radiation sensitivity of the biological activity of purified Bacillus subtilis transforming DNA have been examined. It has previously been shown that the sensitivity of transforming DNA irradiated in dilute solution is less when irradiation is performed in 100 per cent O2 than when in 100 per cent N2, i.e. O2 protects transforming DNA with a dose-modifying factor of about 0.7. DTT protects transforming DNA in a manner that is dependent on DTT concentration and on gassing conditions. In O2 the DTT protection can largely be attributed to the scavenging of .OH radicals by the DTT, but in anoxia DTT exerts a further protective effect which results in an increasing oxygen enhancement ratio (o.e.r.) with increasing DDT concentration to a maximum o.e.r. of about 14 at 2-5 mM DTT. This additional protective effect of DTT is attributable to hydrogen atom donation from DTT to DNA radicals, thus chemically repairing the DNA. Oxygen appears to block this chemical repair reaction.

Bacteria↗
PubMed1984

Effects of oxygen and sulphydryl-containing compounds on irradiated transforming DNA. II. Glutathione, cysteine and cysteamine.

This paper extends our earlier observations on the effects of the sulphydryl (SH)-containing compound dithiothreitol (DTT) on the radiation response of Bacillus subtilis transforming DNA to three other SH-containing compounds-cysteamine, cysteine and glutathione (GSH). In general, all four compounds protect transforming DNA in a manner which is dependent on gassing conditions. In O2, the protection is consistent with the scavenging of OH radicals by the SH compounds, but in N2 there is additional protection which may be due to hydrogen atom donation from the SH compound to radiation-induced DNA lesions, a process which is blocked by O2. This additional protection in N2 results in an increase in the ratio of inactivation in the absence and presence of oxygen with increasing SH concentration to a maximum followed by a decrease at high SH concentrations. The maximum value of the ratio and the SH concentration at which it occurs depend on the SH compound. In particular, GSH appears to be significantly less efficient in the hydrogen-donation repair reaction with transforming DNA than are the other three SH compounds. Furthermore, on the basis of our results, we postulate the existence of a damage fixation process which occurs in the absence of O2, in competition with damage repair by SH compounds, and that this anoxic damage fixation occurs at a rate not less than 300 s-1. We also demonstrate here that the damage fixing reaction of O2 with transforming DNA radicals proceeds 200-fold faster than the competing repair reaction by hydrogen-donation from DTT.

Bacillus subtilis↗
PubMed1988

Transformation of Neurospora crassa with the trp-1 gene and the effect of host strain upon the fate of the transforming DNA.

Neurospora trp-1+ transformants, obtained by transforming a trp-1 inl strain with plasmid DNA containing the wild type trp1+ gene, were characterized by genetic and Southern blot analyses. The transforming trp-1 gene integrated at or near the resident site in all of the trp-1+ transformants obtained with circular DNA or DNA cut within the trp-1 coding region. The frequency of homologous integration decreased substantially when the donor DNA was cleaved outside the trp-1 coding region. The transformants were very stable mitotically and, in general, also showed meiotic stability. Analysis of trp-1+ transformants obtained with another recipient strain, trp-1+ ga-2 aro-9 inl, showed that homologous integration of donor DNA occurred in only 20% of the transformants, whether circular or linear DNA was used. Thus, the host strain employed for transformation appears to be a major factor in determining the fate of transforming DNA. Southern blot analysis of transformants showed that integration of the transforming DNA at the homologous site occurred by double crossover or gene conversion events rather than by insertion of the entire plasmid DNA. Multiple and apparently non functional integration events were observed in some transformants.

DNA Restriction Enzymes↗
PubMed1997

Natural transformation and availability of transforming DNA to Acinetobacter calcoaceticus in soil microcosms.

A small microcosm, based on optimized in vitro transformation conditions, was used to study the ecological factors affecting the transformation of Acinetobacter calcoaceticus BD413 in soil. The transforming DNA used was A. calcoaceticus homologous chromosomal DNA with an inserted gene cassette containing a kanamycin resistance gene, nptII. The effects of soil type (silt loam or loamy sand), bacterial cell density, time of residence of A. calcoaceticus or of DNA in soil before transformation, transformation period, and nutrient input were investigated. There were clear inhibitory effects of the soil matrix on transformation and DNA availability. A. calcoaceticus cells reached stationary phase and lost the ability to be transformed shortly after introduction into sterile soil. The use of an initially small number of A. calcoaceticus cells and nutrients, resulting in bacterial growth, enhanced transformation frequencies within a limited period. The availability of introduced DNA for transformation of A. calcoaceticus cells disappeared within a few hours in soil. Differences in transformation frequencies between soils were found; A. calcoaceticus cells were transformed at a higher rate and for a longer period in a silt loam than in a loamy sand. Physical separation of DNA and A. calcoaceticus cells had a negative effect on transformation. Transformation was also detected in nonsterile soil microcosms, albeit only in the presence of added nutrients and at a reduced frequency. These results suggest that chromosomal DNA released into soil rapidly becomes unavailable for transformation of A. calcoaceticus. In addition, strain BD413 quickly loses the ability to receive, stabilize, and/or express exogenous DNA after introduction into soil.

Acinetobacter calcoaceticus↗
PubMed1996

Mutagenesis and ultraviolet inactivation of transforming DNA of Haemophilus influenzae complexed with a Bacillus subtilis protein that alters DNA conformation.

The wild-type Bacillus subtilis spore protein, SspCwt, binds to DNA in vitro and in vivo and changes the conformation of DNA from B to A. Synthesis of the cloned SspCwt gene in Escherichia coli also causes large increases in mutation frequency. Binding of SspCwt to transforming DNA from Haemophilus influenzae made the DNA resistant to ultraviolet (UV) radiation. The mutant protein, SspCala, which does not bind DNA, did not change the UV resistance. The UV sensitivity of the DNA/SspCwt complex was not increased when the recipients of the DNA were defective in excision of pyrimidine dimers. These data indicate that the H. influenzae excision mechanism does not operate on the spore photoproduct formed by UV irradiation of the complex. Selection for the streptomycin- or erythromycin-resistance markers on the transforming DNA evidenced significant mutations at loci closely linked to these, but not at other loci. SspCwt apparently entered the cell attached to the transforming DNA, and caused mutations in adjacent loci. The amount of such mutations decreased when the transforming DNA was UV irradiated, because UV unlinks linked markers.

Anti-Bacterial Agents↗
PubMed1984

Effects of oxygen and sulphydryl-containing compounds on irradiated transforming DNA. III. Reaction rates.

The absolute rate for the repair reaction of radiation-induced, oxygen-dependent lesions in bacterial transforming DNA with the sulphydryl (SH)-containing compound dithiothreitol (DTT) has been determined using a fast response method, the gas explosion technique, to be 1.6 X 10(6) mol-1 s-1. Glutathione reacts ten times slower than DTT with the irradiated transforming DNA. It can also be calculated that transforming DNA radicals react with O2 in a damage-fixing reaction with a rate of about 3 X 10(8) dm3 mol-1 s-1. These rates are compared with values in the literature for reaction rates of SH, compounds and O2 with irradiated DNA constituents and with bacterial cells.

Bacillus subtilis↗
PubMed1986

The right border region of pTiT37 T-DNA is intrinsically more active than the left border region in promoting T-DNA transformation.

Deletions of border regions of T-DNA on Agrobacterium Ti plasmid or mini-T plasmid have shown the right border region of pTiT37 T-DNA to be more active than the left border region in promoting T-DNA transformation. In this study we examine the possibility that the apparent difference in activity of left and right border regions may be due to position or orientation differences between the left and right borders with respect to the transferred onc genes. We have constructed eight similar single-border mini-T plasmids that contain a left border segment or a right border segment of pTiT37 T-DNA at various positions with either orientation with respect to the onc genes. We assayed the plant tumor-inducing activity of these mini-T plasmids in Agrobacterium tumefaciens LBA4404 containing the virulence helper plasmid pAL4404. Regardless of the position and orientation of the border-containing segment in the mini-T plasmid, mini-T plasmids with the right border segment were highly virulent, whereas those with the left border segment were only weakly so. These results indicate that the difference in transformational activity between the left and right border regions is intrinsic and not an effect of position or orientation with respect to the onc genes. The pattern of the mini-T plasmid sequences integrated into the plant genome suggests that T-DNA transformation involves the directional transfer of a linear intermediate bounded by the border repeats.

Base Sequence↗