Single-stranded regions in Streptococcus pneumoniae chromosomal deoxyribonucleic acid and their relation to transformation.

Deoxyribonucleic acid (DNA) in lysates of both completent and noncompetent streptococcus pneumoniae cells was characterized by chromatography on benzoylated, naphthoylated diethylaminoethyl-cellulose columns, by sensitivity to Aspergillus oryzae S1 endonuclease, and by sucrose gradient analysis. The DNAs from both competent and noncompetent cells were found to contain similar extents of single-stranded regions. These single-stranded regions appeared to be intact, unpaired regions in double-stranded DNA rather than gaps, nicks, or unpaired ends in the DNA. Inhibition of cells with rifampin prior to lysis increased the amount of such single strandedness in the DNA. Lysates made at various times after [14C]thymidine-labeled cells had bound [3H]thymidine-labeled transforming DNA were also characterized by benzoylated, naphthoylated diethylaminoethyl-cellulose chromatography. Changes in the elution profiles of DNA from cells exposed to homospecific (S. pneumoniae) donor DNA were indicative of the formation of complexes between donor DNA and the single-stranded regions of recipient DNA. In contrast, profiles of DNA from cells exposed to heterospecific (S. sanguis) DNA did not show significant changes, indicating that few such donor-recipient complexes were formed during heterospecific transformation.     

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.     

Competence Mutant of Haemophilus influenzae with Abnormal Ratios of Marker Efficiencies in Transformation

In studies of competence-deficient mutants of Haemophilus influenzae which absorb deoxyribonucleic acid (DNA) but fail to produce transformants, it was observed that in some mutants the residual transforming activity for different markers varied widely, i.e., produced a ratio effect. One of these mutants, com⁻⁵⁶, was studied intensively to determine the cause of the residual efficiency of transformation and the reason for the ratio effect. The residual frequency of transformation was higher for markers considered single-site mutations (like naladixic acid resistance), whereas the least efficient markers tested were those conferring resistance to high levels of streptomycin or novobiocin which are more complex than single-site mutations. Measurement of frequencies of cotransformation indicated that overall genetic linkage was reduced. Transfection was fairly efficient with phage S2 DNA, but not prophage DNA. Donor marker activity could be detected in transformed cell lysates, but not linked to recipient markers in recombinant molecules. Sucrose gradient analysis of such lysates revealed that donor material was associated with recipient DNA in at least normal quantities, but lacked detectable genetic activity. Material from donor DNA labeled with heavy isotopes was incorporated into recipient chromosomal fragments having a density indistinguishable from normal density, unlike the hybrid density recombinant material found in normal cells. No excessive solubilization or nicking of unincorporated donor was detected. It is postulated that this strain contains a hyperactive nuclease, which reduces the effective size of the input DNA during the integration process.     

Transformation in Bacillus subtilis: characterization of an intermediate in recombination observed during transformation.

Summary From recombination-proficient competent cells of Bacillus subtilis in which the donor DNA entered at 17°, and which were kept at the same temperature, a complex of donor DNA and the recipient chromosome can be obtained which has a relatively high buoyant density in CsCl gradients. Exposure of the isolated complex to nuclease S1 liberates donor radioactivity. The limited biological activity of DNA re-extracted from cells attempting to recombine at 17° is decreased upon incubation with nuclease S1. If recombination is allowed to proceed at 30°, the high buoyant density of the donor-recipient complex decreases to normal values and less radioactivity can be liberated from the complex by nuclease S1. Concomitantly the biological activity of re-extracted DNA becomes less vulnerable to nuclease S1 under these conditions. On the basis of these observations we assume that the intermediate complex partly consists of unpaired single-stranded donor DNA.Support for the correctness of this assumption is derived from experiments with a mutant, which is delayed in the processing of high buoyant density donor-recipient complex to normal buoyant density donor-recipient complex. This delay is reflected in the time of acquisition of resistance to nuclease S1 digestion of the isolated complex.     

Discontinuous DNA replication in mouse P-815 cells.

The length of newly synthesized DNA strands from mouse P-815 cells was analyzed after denaturation both by electrophoresis and by sedimentation in alkaline sucrose gradients. [3-H]-Thymidine pulses of 2-8 min at 37 degrees C predominantly label molecules of 20-60 S. With 30-s pulses at 25 degrees C, all the [3-H]thymidine appears in short DNA strands of 50-200 nucleotides. Thus, DNA strand elongation occurs discontinuously via Okazaki fragments at both the 5' end and the 3' end. In dodecylsulfate lysates, only 10% of the Okazaki fragments are found as single-stranded molecules. About 90% are resistant to hydrolysis by the single-strand-specific nuclease S-1 and band in isopycnic gradients at the buoyant density of double-stranded DNA. No evidence for ribonucleotides at the 5' end of Okazaki fragments was obtained either in isopycnic CsCl or Cs2SO4 gradients or after incubation with polynucleotide kinase and [gamma-32P]ATP.     

Single-stranded regions in DNA isolated from different developmental stages of the sea urchin

Long-term labeled sea urchin embryo (Strongylocentrotus purpuratus) DNAs were examined for size of recovered pieces, single-strandedness, and length of continuous double-stranded regions. Sizing on neutral sucrose gradients indicates that morula stage DNA sediments predominantly at 31 S, blastula stage DNA at 27 S, and gastrula stage DNA as a broad range of sizes of greater than 29 S. Treatment of [3H]thymidine-labeled DNA with Aspergillus oryzae S1 nuclease removes 19% of the 3H from morula stage DNA, 4% of the 3H from blastula stage DNA, and less than 0.1% of the 3H from gastrula stage DNA. Sedimentation of S1 nuclease treated [3H]DNAs on alkaline sucrose gradients indicates that in native morula stage DNA there is a nick or gap in one strand approximately every 9700 base pairs, in native blastula stage DNA about every 3300 base pairs, and very few nicks or gaps in native gastrula stage DNA.     

Fate of Transforming Deoxyribonucleate in Bacillus subtilis

The majority of donor deoxyribonucleate (DNA) at early stages after uptake was found in a complex with a cell component which changes its buoyant behavior on equilibrium density gradients. Analysis of the recipient cell lysates, after treatment to dissociate the complex, showed about two-thirds of the donor molecules in denatured form and the rest associated with recipient DNA. Incubation of cells after DNA uptake leads to the disappearance of denatured donor DNA and to the increase of donor label associated with recipient DNA. Some characteristics of a component from intact cells or spheroplasts with affinity for denatured Bacillus subtilis DNA are described.     

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.     

Cross-links in African swine fever virus DNA.

African swine fever virus DNA sediments in neutral sucrose density gradients as a single component with a sedimentation coefficient of 60S. In alkaline sucrose density gradients, this material shows two components with sedimentation coefficients of 85S and 95S, respectively. The sedimentation rate value of alkali-denatured virus DNA in neutral sucrose density gradients and the renaturation velocity of denatured DNA show that is reassociated much faster than expected from its genetic complexity. This behavior is compatible with the existence of interstrand cross-links in the molecule. We also present results which suggest that there are only a few such cross-links per molecule, that they are sensitive to S1 nuclease digestion, and that they are probably located next to the ends of the DNA.     

Characterization of large mammalian DNA species sedimented in a reorienting zonal rotor

The characteristics of a Beckman-designed slow acceleration unit for the reorientation of alkaline sucrose gradients in a Ti-15 zonal rotor are described. The large DNA species (> 250S) obtained from cultured rat brain tumor cells with this system sediment linearly with time, have virtually no [³H]leucinelabeled or covalently bonded [³H]choline-labeled material sedimenting with them, sediment independently of smaller single-stranded DNA molecules (? 165S) and are 60–80% degraded by the single-strand-specific S1 nuclease. Therefore, it is postulated that these species are collapsed, partially denatured DNA molecules or a collapsed form of single-stranded DNA. When cells were labeled with [¹⁴C]TdR, then frozen and stored at ? 79°C, this system could detect radiation-induced DNA damage from decay of the incorporated label at accumulated doses as small as 18–126 rads.     

Characteristics of a complex formed by a nonintegrated fraction of transforming DNA and Bacillus subtilis recipient cell constituents.

Summary Nonintegrated transforming DNA was isolated from recipient cell lysates as a complex with cellular constituents (natural complex) and separated from free proteins on CsCl density gradients. Sensitivity of DNA in this complex to digestion with endonuclease S₁, liberation of denatured donor molecules by treatment of the cell lysates with phenol, as well as previously described liberation of single-stranded donor DNA by heating with detergents, pointed to the single-stranded nature of the donor DNA in the complex. About 1% of radioactive leucine or phenylalamine incorporated to cellular proteins were detected in the natural complex, with two associated enzymatic activities: one autolytic, the other endonucleolytic. The autolytic activity, known to be localized mainly in the cell wall and the endonucleolytic one, similar to the enzyme localized in cell membrane and periplasmic space of B. subtilis, suggest that donor DNA is complexed with a cell wall and/or a cell membrane fragment. Consideration of several characteristics of the natural complex: its density, protein content, and partial resistance of its DNA to DNase I, point to partial shielding of donor DNA in the cell fragment structure, and existence of a portion in a free uncovered form. Considerations on the possible role of the two enzymatic activities were based on the fact that they were not found in the complex formed by denatured DNA added to cells before lysis (reconstruction complex), and on hypotheses of their possible physioligical role.Part of the above results was presented in preliminary form at the Third European Meeting on Bacterial Transformation and Transfection, Granada, Spain, August 31–September 3, 1976     

Translocation of the pre-synaptic complex formed upon DNA uptake by Streptococcus sanguis and its inhibition by ethidium bromide.

Summary Donor DNA in its initially bound, singlestranded form exists in a chromosomally-unassociated complex where it is resistant to exogenous DNase I but sensitive to micrococcal nuclease. Most of the complexes are readily recuperable from the supernatant of recipients converted into spheroplasts. Subsequent to formation of this superficially located complex, donor DNA progressively associates with the recipient chromosome into which it is eventually integrated. Treatment of recipients with ethidium bromide at various times after initial DNA binding almost immediately halts translocation of whatever donor material is not yet synapsed with the chromosome. On the other hand, donor DNA that has already synapsed experiences no difficulty in becoming genetically integrated. Some degradation occurs to DNA that fails to undergo translocation as a result of ethidium bromide treatment, the acid-soluble products appearing in the culture medium. DNA in untranslocated complexes surviving treatment is not appreciably different in single-strand length from that in untreated complexes. When these surviving complexes are isolated from a cell lysate, the contained DNA can be shown by spectrofluorometry to have bound the drug.     

Analysis of the distribution of DNA repair patches in the DNA-nuclear matrix complex from human cells

The distribution of ultraviolet-induced repair patches along DNA loops attached to the nuclear matrix, was investigated by digestion with DNA-degrading enzymes and neutral sucrose gradient centrifugation. When DNA was gradually removed by DNAase 1, pulse label incorporated by ultraviolet-irradiated cells during 10 min in the presence of hydroxyurea or hydroxyurea/arabinosylcytosine showed similar degradation kinetics as prelabelled DNA. No preferential association of pulse label with the nuclear matrix was observed, neither within 30 min nor 13 h after irradiation. When the pulse label was incorporated by replicative synthesis under the same conditions, a preferential association of newly-synthesized DNA with the nuclear matrix was observed. Single-strand specific digestion with nuclease S1 of nuclear lysates from ultraviolet-irradiated cells, pulse labelled in the presence of hydroxyurea/arabinosylcytosine, caused a release of about 70% of the prelabelled DNA and 90% of the pulse-labelled DNA from the rapidly sedimenting material in sucrose gradients. The results suggest no specific involvement of the nuclear matrix in repair synthesis, a random distribution of repair patches along the DNA loops, and simultaneously multiple incision events per DNA loop.     

Effect of 4,5'',8-trimethylpsoralen interstrand cross-links present in recipient Bacillus subtilis on the integration of transforming DNA.

When recipient Bacillus subtilis carrying chromosomal trimethylpsoralen cross-links were transformed, the donor marker activity decreased with the extent of cross-linking. Additional donor marker activity was lost upon incubation of the reextracted DNA with nuclease S1, particularly at higher levels of cross-linking. Physical analysis of the reextracted DNA showed that the donor DNA was progressively excluded from heteroduplex formation as the frequency of cross-links in the recipient DNA increased. In the donor-recipient complexes still being formed, increasing amounts of donor DNA became susceptible to nuclease S1 digestion under these conditions. These results suggest that resident interstrand cross-links interfere both with initiation of recombination and with the completion of heteroduplex formation.     

Mechanism of conjugation and recombination in bacteria. XVII. Further evidence for single-stranded regions in recipient DNA during conjugation in Escherichia coli K-12.

The secondary structure of recipient DNA mated with Hfr strain was investigated by CsCl density gradient fractionation. After 45 min of HfrH64 X 3h-f-ab1157 mating one-fourth of the radioactive recipient DNA was recovered as a single-strand but only after shearing of cell lysates prior to centrifugation. This heavier than native DNA fraction of radioactive material (obtained after the first centrifugation) was degraded by single-strand specific nuclease S from Aspergillus oryzae. These findings thus confirm the authors' earlier results suggesting that in the course of mating are generated local single-stranded regions in recipient DNA.     

Relation of Macromolecular Synthesis in Streptococci to Efficiency of Transformation by Markers of Homospecific and Heterospecific Origin

In previous studies with Streptococcus sanguis and S. pneumoniae as recipients and donors of transforming deoxyribonucleic acid (DNA), it was found that heating recipients just prior to exposure to DNA caused an increase in the number of transformants induced by heterospecific DNA relative to that induced by homospecific DNA. In the present studies, S. sanguis recipients were found to recover from this effect of heat (48 C, 15 min) when incubated at 37 C before exposure to DNA. Inhibitors of nucleic acid synthesis, such as rifampin, 5-fluorodeoxyuridine, actinomycin, and p-hydroxyphenylazo-uracil, but not inhibitors of protein synthesis, such as chloramphenicol and erythromycin, prevented recovery from the effect of heat. Inhibitors of nucleic acid synthesis caused changes in unheated cells similar to those observed with heat treatment; these changes included increased transformability by genetically hybrid DNA and by low-efficiency markers in homospecific DNA. The effect of a combination of heat and inhibitors on transformation by heterospecific DNA was greater than when single treatments were used. The most effective inhibitor used alone was rifampin: in treated recipient cells, the yield of transformants produced by a given amount of irreversibly bound heterospecific DNA was increased without a significant change in the yield of transformants produced by bound homospecific DNA. A cell being doubly transformed by homospecific and heterospecific DNA was enhanced specifically in its transformability with the latter as a consequence of rifampin treatment. Treatment with rifampin also increased co-transformation by linked heterospecific markers. The period during which recipient cells were sensitive to the effects induced by rifampin and fluorodeoxyuridine lasted from 10 to 20 min after DNA uptake.     

Studies on superinfection immunity among transmissible plasmids in Escherichia coli

Superinfection immunity was studied by a method which permits the specific labeling of plasmid DNA following its entry into a recipient cell during conjugation. By measuring the incorporation of [³H]thymine during matings between a donor strain of Escherichia coli K12 carrying the R factor, Rl, and various recipients, we found that the presence in the recipient of a plasmid closely related to R1 (F or R factor 222), or isogenic to it (resistance transfer factor, from Rl), resulted in a reduction of 80 to 90% in the rate of [³H]thymine incorporation, relative to a mating with a plasmid-negative (F ^?) recipient. The DNA present in these recipients after 60 minutes of mating was further examined by neutral sucrose gradient eentrifugation. The DNA in the F⁺ and F ^? recipients sedimented similarly, in two major peaks at 50 S (relaxed circles) and 75 S (supercoiled circles). However, the DNA in the RTF recipient sedimented at rates intermediate between 50 S and 75 S. Pulse-chase experiments revealed that the DNA species seen after 60 minutes of an R1 × RTF mating are normal replicative intermediates which have disappeared by 60 minutes in the R1 × F^? or R1 × F⁺ matings.These data support genetic evidence suggesting that superinfection immunity is due to two distinct effects—entry exclusion and plasmid incompatibility. Thus, F (related to R1 but genetically compatible with it), as well as the incompatible plasmids, 222 and the RTF of R1 itself, when present in the recipient, greatly reduce the total synthesis of newly introduced R1 DNA in the recipient. We interpret this effect as entry exclusion. Incompatibility, manifested by RTF, but not F, further reduces the efficiency of conjugation by slowing the rate at which a newly acquired plasmid is replicated.     

Molecular Fate of Heterologous Bacterial DNA in Competent BACILLUS SUBTILIS. II. Unstable Association of Heterologous DNA with the Recipient Chromosome

In CsCl density gradients of lysates from competent Bacillus subtilis cells, which had been exposed to heterologous bacterial DNA, very little donor-recipient complex (DRC) formation could be detected. The present study demonstrates that photocrosslinking of such lysates by irradiation with long-wave UV light in the presence of 4,5',8-trimethylpsoralen results in a dramatic increase in the amount of heterologous DRC. This phenomenon may be interpreted as the stabilization of a pre-existing weak association between entered heterologous donor DNA and one recipient strand in unpaired regions of the chromosome. When a recombination-deficient mutant is used, the amount of stabilizable heterologous DRC is reduced to the same extent as the specific transforming activity of homologous DNA. Although the amount of stabilizable complex is related to the degree of homology between donor and recipient DNA, this relation is not a quantitative one. Probably the association is caused by very short regions of base pairing between the donor and recipient moieties in the complex. Heating of a lysate at 70° prior to photocrosslinking prevents stabilization, apparently because the regions of base pairing are rapidly melted out. The results described in this paper can be best interpreted as the fixation of a process in which entered donor DNA in competent cells tries to find homologous stretches in the recipient chromosome.     

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis: further characterization of unstable association between donor and recipient DNA and the involvement of the cellular membrane

Summary Although heterospecific transformation is extremely inefficient and very little heterologous donor DNA integrates into the recipient chromosome in a stable way, we have previously shown that B. pumilus DNA entering competent B. subtilis efficiently associates with the recipient chromosome in an unstable way. This association can be stabilized by photocrosslinking in the presence of 4,5,8-trimethylpsoralen; it depends on the recombination proficiency of the recipient strain and on strand-separation of the recipient chromosome (te Riele and Venema 1982b). The present study provides further evidence that the heterologous donor DNA and the recipient DNA are associated by regions of base-pairing. Based on the high sensitivity of the donor moiety in the complex to nuclease S1 (90%) and the high sensitivity of the complex to moderate denaturing conditions (Tm=48°C), we presume that donor and recipient DNA are associated either by several short sequences of 15–25 fairly well matched base pairs or by a region of base-pairing of about 200 bases, which contains 25% of mismatches. During incubation, the unstable complex disappears, probably due to nucleolytic degradation.The unstable heterologous donor-recipient complex (DRC) was found to be membrane-bound. However, in contrast to homologous DRC, the unstable heterologous DRC remains membrane bound during incubation. Apparently, the predominantly single-stranded character of the heterologous DRC prevents release of the complex from the membrane.Abbreviations DRC donor-recipient complex - TMP 4,5,8-trimethyl-psoralen - DNAase I deoxyribonuclease 1 - TCA trichloroacetic acid     

Surface-located trypsin-activated Streptococcus sanguis strain Wicky endonuclease

A new Streptococcus sanguis strain Wicky endonuclease was isolated, purified and partially characterized. This nuclease acts preferentially on thermally denatured DNA, is not inhibited by RNA and is activated 3-5 times by trypsin. This activation is accompanied by the reduction of molecular weight of the enzyme. These features distinguish the new S, sanguis nuclease from the 3 previously described S. sanguis endonucleases. With covalently closed circular plasmid DNA, the enzyme causes first the appearance of a single stranded nick, then the second nick on the opposite DNA strand, resulting in plasmid DNA linearization. This nuclease most likely is located at the cell surface. The possible relationship of the described nuclease with ability of S. sanguis cells to take up DNA in genetic transformation is discussed.