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.     

Repair of thermal damage to the Escherichia coli nucleoid.

The folded chromosome or nucleoid of Escherichia coli was analyzed by low-speed sedimentation in neutral sucrose gradients after heat treatment (30 min at 50 degrees C) and subsequent incubation of cells at 37 degrees C for various times. Heat treatment resulted in in vivo association of the nucleoids with cellular protein and in an increase in sedimentation coefficient. During incubation at 37 degrees C, a fraction of the nucleoids, from heated cells, because dissociated from cellular protein and regained their characteristic sedimentation coefficients. The percentage of nucleoids which returned to their control sedimentation position in the sucrose gradients corresponded to the percentage of cells able to repair thermal damage as assayed by enumeration on agar plates.     

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.     

Studies on the compaction of isolated nucleoids from Escherichia coli

The genomic DNA of Escherichia coli is contained in one or two compact bodies known as nucleoids. Isolation of typically shaped nucleoids requires control of DNA expansion, accomplished here by a modification of the polylysine-spermidine procedure. The ability to control expansion of in vitro nucleoids has application in nucleoid purification and in preparation of samples for high-resolution imaging, and may allow an increased resolution in gene localization studies. Polylysine of relatively low average molecular weight (approximately 3 kDa) is used to produce lysates containing nucleoids that are several-fold expanded relative to the sizes of in vivo nucleoids. These expanded forms can be converted to compact forms similar in dimensions to the cellular nucleoids by either a further addition of polylysine or by incubation of diluted lysates at 37 degrees C. The incubation at 37 degrees C is accompanied by autolytic degradation of most ribosomal RNA. Hyperchromism and circular dichroism spectra indicate that polylysine-DNA complexes are modified during the incubation. Compact forms of the nucleoid can be progressively reexpanded by exposure to salt solutions. Nucleoid compaction was similar in lysates made from rapidly or slowly growing cells or from cells that had been briefly treated with chloramphenicol to reduce linkages between DNA and cell envelope.     

Inactivation of transforming Bacillus subtilis deoxyribonucleic acid by monoadducts of 4,5',8-trimethylpsoralen.

4,5',8-Trimethylpsoralen (TMP) monoadducts inactive transforming deoxyribonucleic acid (DNA) in Bacillus subtilis. Contrary to TMP diadducts (TMP cross-links), which severely inhibit entry of donor DNA (G. Venema and U. Canosi, Mol. Gen. Genet. 179:1--11), TMP monoadducts have only a slight effect on entry. Since reextracted TMP-monoadduct-containing transforming DNA is a differentially repaired by Uvr- and Uvr+ recipients and cross-linkable to the recipient strand in the heteroduplex recombinant DNA molecules, the monoadducts can be integrated along with the donor DNA into the recipient chromosome.     

Effect of ethylenediaminetetraacetic acid on deoxyribonucleic acid entry and recombination in transformation of a wild-type strain and a rec-1 mutant of Haemophilus influenzae

In transformation of Haemophilus influenzae, donor deoxyribonucleic acid (DNA) enters into competent cells in the presence of ethylenediaminetetraacetic acid (EDTA), which prevents the formation of single stranded regions in the donor DNA that has entered. If after entry of DNA the recipient cells were first incubated at 17 degrees C and then at 37 degrees C in the continuous presence of EDTA, almost no integration occurred. On the other hand, if after entry of DNA the cells were incubated first at 17 degrees C in the absence of EDTA, allowing the generation of single-stranded regions (integration is blocked at this temperature), and then at 37 degrees C in the presence of EDTA, donor-recipient DNA complexes were formed. These results suggest that single-stranded regions are required for integration. Integration to completion was strongly inhibited by EDTA. In a rec-1 mutant of H. influenzae no donor-recipient DNA complexes carrying recombinant-type activity were formed during incubation at 37 degrees C in the absence of EDTA. If rec-1 cells were incubated at 37 degrees C in the presence of EDTA, which strongly inhibited breakdown of DNA, donor-recipient DNA complexes were formed if previously single-stranded regions in the donor DNA that had entered were generated by incubation at 17 degrees C in the absence of EDTA. This suggests that the rec-1 protein protects the initial donor-recipient DNA complex against degradation, so that further steps in the recombination process can proceed.     

Intermediates in the formation of the open complex by RNA polymerase holoenzyme containing the sigma factor sigma 32 at the groE promoter

The interaction of E sigma 32 with the groE promoter at temperatures between 0 degrees C and 37 degrees C was studied using DNase I footprinting and dimethyl sulfate methylation. Three distinct complexes were observed. At 0 degrees C E sigma 32 fully protected sequences between -60 and -5 from DNase I digestion on the top (non-template) strand of the promoter. At 16 degrees C the majority of the E sigma 32 promoter complexes had a DNase I footprint almost identical with that seen at 37 degrees C, protecting the DNA from about -60 to +20; however, little DNA strand separation had occurred, and the changes in sensitivity of guanine residues to dimethyl sulfate methylation caused by E sigma 32 differed from those seen at 37 degrees C. DNA strand separation, and changes in the pattern of protections from and enhancements of methylation by dimethyl sulfate to those characteristic of the open complex, occurred at temperatures between 16 degrees C and 27 degrees C. It is plausible to assume that these temperature-dependent isomerizations are analogous to the time-dependent sequence of intermediates on the pathway to open complex formation at 37 degrees C. Therefore we propose that the formation of an open complex by E sigma 32 at the groE promoter involves three classes of steps: E sigma 32 initially binds to the promoter in a closed complex (RPC1) in which the enzyme interacts with a smaller region of the DNA than in the open complex. E sigma 32 then isomerizes to form a second closed complex (RPC2) in which the enzyme interacts with the same region of the DNA as in the open complex. Finally, a process of local DNA denaturation (strand opening) leads to formation of the open complex (RPO).     

Folding of prokaryotic DNA. Isolation and characterization of nucleoids from Bacillus licheniformis

Intact and fast-sedimenting nucleoids of Bacillus licheniformis were isolated under low-salt conditions and without addition of detergents, polyamines or Mg2+. These nucleoids were partially unfolded by treatment with RNase and completely unfolded by treatments that disrupt protein-DNA interactions, like incubation with proteinase K, 0.1% sodium dodecyl sulphate and high ionic strength. Ethidium bromide intercalation studies on RNase-treated, proteinase-K-treated and non-treated nucleoids in combination with sedimentation analysis of DNase-I-treated nucleoids revealed that DNA is organized in independent, negatively supertwisted domains. In contrast to the DNA organization in bacterial nucleoids, isolated under high-salt conditions and in the presence of detergents (Stonington & Pettijohn, 1971; Worcel & Burgi, 1972), the domains of supertwisted DNA in the low-salt-isolated nucleoids studied here are restrained by protein-DNA interactions. A major role for nascent RNA in restraining supertwisted DNA was not observed. The superhelix density of B. licheniformis nucleoids calculated from the change of the sedimentation coefficient upon ethidium bromide intercalation, was of the same order of magnitude as that of other bacterial nucleoids and eukaryotic chromosomes, isolated under high-salt conditions: namely, -0.150 (corrected to standard conditions: 0.2 M-NaCl, 37 degrees C; Bauer, 1978). Electron microscopy of spread nucleoids showed relaxed DNA and regions of condensed DNA. Spreading in the presence of 100 micrograms ethidium bromide per ml revealed only condensed structures, indicating that nucleoids are intact. From spreadings of proteinase-K-treated nucleoids we infer that supertwisted DNA and the protein-DNA interactions, responsible for restraining the superhelical DNA conformation, are localized in the regions of condensed DNA.     

Unstable association in vitro between donor and recipient DNA in Bacillus subtilis.

In addition to stable donor-recipient DNA complexes, unstable complexes between donor and recipient DNA were formed in vitro with Bacillus subtilis. Whereas the stable complexes survived CsCl gradient centrifugation at pH 11.2 and phenol plus sodium p-aminosalicylate extraction with 0.17 M NaCl, the unstable complexes dissociated during these manipulations. The donor moiety from the unstable complexes remained associated with the recipient DNA during phenol plus sodium p-aminosalicylate treatment at 0.85 M NaCl. The unstable complexes could be stabilized artificially by cross-linking with 4,5',8-trimethylpsoralen. Dissociation of the complexes during CsCl gradient centrifugation could be prevented by centrifuging at pH 10. Heterologous DNA fragments derived from phage H1 DNA appeared to be unable to form complexes with the recipient B. subtilis DNA. Unstable complexes were also formed with Escherichia coli DNA, although under all conditions tested, more complex was detectable by using homologous B. subtilis DNA.     

Alkali lability and rapid initiation of excision repair following photoaffinity damage by ethidium azide

DNA damage and repair provoked by ethidium azide (EA) photoaffinity labeling in mouse leukemia cells was studied by measuring sedimentation properties of nucleoids in neutral sucrose gradients, and it was found that the strand opening step was faster than that which followed damage of cells by ultraviolet (UV) light. The two insults were compared at levels of damage which gave the same overall rates of repair synthesis in intact cells and which required the same length of time to complete repair, as judged by the restoration of supercoiling of the isolated nucleoids. In the case of UV, single-strand breaks in DNA were detectable at 30 min, maximum at 2 h, and the superhelical properties restored at 21 h. With photoaffinity labeling, single-strand breaks were prominent immediately, even when photolabeling of cells was done on ice, but restoration of DNA supercoiling still required 21 h. Photolabeling of isolated nucleoids or isolated viral DNA with EA failed to introduce DNA strand breaks. However, it was discovered that photoaffinity labeling of DNA with EA resulted in alkali labile sites shown by single strand breaks produced on alkaline sucrose sedimentation or by alkali exposure followed by sedimentation on neutral formamide gradients. These results suggest that the drug attachment sites should be identifiable by the location of such single strand breaks.     

Chemical radiosensitization by misonidazole: production and repair of DNA single-strand breaks in Yoshida ascites tumor cells.

Formation of strand-breaks in DNA and its repair in Yoshida ascites tumor cells exposed to gamma radiation (100-400 Gy) in presence and absence of misonidazole (10 mM) were studied. The methodology involved pre-labelling of cellular DNA by 3H-thymidine during cell proliferation in rats, irradiation of cells in vitro and analysing sedimentation profile of DNA by ultracentrifugation in alkaline sucrose density gradients. Irradiation under euoxic conditions resulted in formation of about 1.5 times greater number of strand breaks as compared to those formed during irradiation under hypoxic conditions. Misonidazole (10 mM) by its presence along with the cells during irradiation under hypoxic conditions caused a 3-fold increase in the number of single strand breaks, but under euoxic conditions of irradiation the presence of misonidazole did not enhance the strand break formation. Incubation of cells irradiated in absence of misonidazole for 1 hr in tissue culture medium at 37 degrees C resulted in repair of substantial fraction of the strand breaks while there was no repair of the DNA strand breaks in cells irradiated in the presence of the chemical.     

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.     

High frequency conjugative mobilization in natural strains of Bacillus subtilis, bearing a large plasmid]

Conjugative properties of the strain Bacillus subtilis that carrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described. The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis 168 strain with a frequency of approximately 10(-2). The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium. The latter fact makes this system suitable as a model for studying conjugal mobilization in bacilli. A large plasmid cannot be transferred to recipients. An optimal temperature for conjugation of donor and recipient cells was 37 degrees C, but conjugation also proceeded at lower temperatures, up to 21 degrees C.     

Differential and dynamic localization of topoisomerases in Bacillus subtilis

Visualization of topoisomerases in live Bacillus subtilis cells showed that Topo I, Topo IV, and DNA gyrase differentially localize on the nucleoids but are absent at cytosolic spaces surrounding the nucleoids, suggesting that these topoisomerases interact with many regions of the chromosome. While both subunits of Topo IV were uniformly distributed throughout the nucleoids, Topo I and gyrase formed discrete accumulations, or foci, on the nucleoids in a large fraction of the cells, which showed highly dynamic movements. Three-dimensional time lapse microscopy showed that gyrase foci accumulate and dissipate within a 1-min time scale, revealing dynamic assembly and disassembly of subcellular topoisomerase centers. Gyrase centers frequently colocalized with the central DNA replication machinery, suggesting a major role for gyrase at the replication fork, while Topo I foci were frequently close to or colocalized with the structural maintenance of chromosomes (SMC) chromosome segregation complex. The findings suggest that different areas of supercoiling exist on the B. subtilis nucleoids, which are highly dynamic, with a high degree of positive supercoiling attracting gyrase to the replication machinery and areas of negative supercoiling at the bipolar SMC condensation centers recruiting Topo I.     

Involvement of single-strand breaks in complex formation between single-stranded DNA and nucleoids of Bacillus subtilis

Summary RNase-unfolded chromosomes of competent Bacillus subtilis are able to take up single-stranded homologous donor DNA fragments in vitro to form donor-recipient DNA complexes (Van Randen and Venema 1981). The unfolded chromosomes behave as supercoiled DNA molecules. X-irradiation increased the formation of unstable and stable complexes between donor and recipient DNA during incubation at 37° C. The complex-forming ability of the unfolded chromosomes increased linearly with increasing X-ray dose, even after complete relaxation of the unfolded chromosomes had occurred. Limited DNase I action increased the complex-forming ability of the chromosomes as effectively as X-irradiation.Unstable donor-recipient DNA complexes can be distinguished from stable ones by their dissociation upon density gradient centrifugation in CsCl at pH 11.2. They are stable at pH 10 (Van Randen et al. 1982a). At an intermediate pH value during isopycnic centrifugation, a fraction of the unstable complexes were stable, suggesting that a range of stabilities existed among the unstable complexes. The donor moiety of the stable donor-recipient DNA complexes was far more resistant to nuclease S1 treatment than that of the unstable ones.     

Single-stranded regions in transforming deoxyribonucleic acid after uptake by competent Haemophilus influenzae.

About 15% of donor deoxyribonucleic acid (DNA) is single stranded immediately after uptake into competent Haemophilus influenzae wild-type cells, as judged by its sensitivity to S1 endonuclease. This amount decreases to 4 to 5% by 30 min after uptake. Mutants which are defective in the covalent association of recipient and donor DNA form little or no S1 endonuclease-sensitive donor. At 17 C donor DNA taken up by the wild type contains single-stranded regions although there is no observable association, either covalent or noncovalent. The single-stranded regions are at the ends of donor DNA molecules, as judged by the unchanged sedimentation velocity after S1 endonuclease digestion. The amount of single-stranded donor remains constant at 17 C for more than 60 min after uptake, suggesting that the decrease observed at 37 C is the result of association of single-stranded ends with single-stranded regions of recipient cell DNA. Three sequential steps necessary for the integration of donor DNA into recipient DNA are proposed: the synthesis of single-stranded regions in recipient DNA, the interaction of donor DNA with recipient DNA resulting in the production of single-stranded ends on donor DNA, and the stable pairing of homologous single-stranded regions.     

Fate of homospecific transforming DNA bound to Streptococcus sanguis.

The fate of [3H]DNA from Streptococcus sanguis str-r43 fus-s donors in [14C]S. sanguis str-s fus-r1 recipients was studied by examining the lysates prepared from such recipients at various times after 1 min of exposure to DNA. The lysates were analyzed in CsCl and 10 to 30% sucrose gradients; fractions from the gradients were tested for biological activity and sensitivity to nucleases, subjected to various treatments and retested for nuclease sensitivity, and run on 5 to 20% neutral and alkaline sucrose gradients. The results demonstrate that donor DNA bound to S. sanguis cells in a form resistant to exogenous deoxyribonuclease is initially single stranded and complexed to recipient material. Donor DNA can be removed from the complex upon treatment of the complex with Pronase, phenol, or isoamyl alcohol-chloroform. Within the complex, donor DNA is relatively insensitive to S1 endonuclease but can regain its sensitivity by treatment with phenol. With time the complex moves as a whole to associate physically with the recipient chromosome. After a noncovalent stage of synapsis, donor material is covalently bonded to and acquires the nuclease sensitivity of recipient DNA, while donor markers regain transforming activity and become linked to resident markers.     

The integration of donor and recipient deoxyribonucleic acid during transformation of Bacillus subtilis

1. DNA labelled with 5-bromo[(3)H]uracil was used to transform auxotrophic strains of Bacillus subtilis. 2. After various times of incubation, DNA was extracted from the transformed culture and subjected to equilibrium sedimentation in caesium chloride gradients. 3. In addition to heavy donor DNA and light recipient DNA, a component with an intermediate density was found and is believed to consist of a biological hybrid of donor and recipient. 4. The component of intermediate density was isolated and found to possess activity in transformation derived from both donor and recipient strains. 5. Denaturation of the component of intermediate density followed by centrifugation gave only one component, indicating that integration had occurred in both strands of the recipient DNA. 6. No integrated band was observed after uptake by competent cells of B. subtilis of heavy DNA prepared from Escherichia coli.     

Effect of adriamycin and hyperthermia on the sedimentation of nucleoids from L1210 cells

We report on the individual and combined effects of adriamycin (ADR) and hyperthermia (HYP) on the sedimentation behavior of L1210 mouse leukemia cell nucleoids in neutral sucrose gradients. Nucleoid sedimentation profiles obtained from cells incubated with ADR (1-10 microM; 30 min; 37 degrees C) exhibited an increased sedimentation rate associated with an increased protein content of these subnuclear units. Exposure of cells to HYP (1-3 h; 42 degrees C) produced similar results. Simultaneous exposure of L1210 cells to conditions of HYP and ADR which resulted in minimal changes in nucleoid sedimentation when used singly, produced an enhanced effect. A similar enhancement was observed with other intercalating antineoplastic agents believed to exert their effect, at least partially, via free radicals (daunorubicin, amsacrine, bisantrene, mitoxantrone). However, enhancement with HYP was not observed with (a) the classic intercalating agent, ethidium bromide; (b) non-intercalating DNA-breaking agents (bleomycin, lithocholic acid, etoposide); (c) inhibitors of poly (ADP-ribose) polymerase (m-methoxybenzamide, benzamide); or (d) non-intercalating antineoplastic agents capable of causing free radical formation (bleomycin). The results suggest that DNA intercalating agents capable of initiating free radical processes may show an enhanced toxicity with simultaneous HYP treatment, and that the nucleoid assay may be a means of screening agents with these biological properties for potential clinical usefulness in combination with HYP.