Heterologous deoxyribonucleic acid uptake and complexing with cellular constituents in competent Bacillus subtilis.

With competent cultures of Bacillus subtilis the uptake of Escherichia coli deoxyribonucleic acid (DNA) is about 50% that for homologous DNA. Uptake of phage T6 DNA, if any, is of the order of 7%, while nonglucosylated phage T6 (T6) DNA is taken up almost as effectively as homologous DNA. Both T6 and T4 DNA interfere only minimally with uptake of homologous DNA; by contrast, T6 DNA competes with homologous DNA as effectively as the latter itself. These results indicate that the glucose residues in the T-even phage DNA, located in the large groove of the DNA helix, reduce affinity for cellular receptors, leading to low binding of T6 DNA. The latter DNA is considerably less degraded by extracellular nucleases than homologous DNA, thus excluding enzymatic hydrolysis as the source of poor uptake. Affinity of DNA for competent cells was also evaluated by the formation, and detection in a CsCl density gradient, of complexes of DNA with cellular constituent(s). Such comlexes, similar to those previously observed with transforming DNA, are formed by E. coli DNA and T6 DNA; in reconstruction experiments the denatured forms of these same DNA samples form complexes when added to the cells before lysis. T6 DNA, on the other hand, does not form such a complex. The possible role of such complexes in transport of DNA to the cell interior is discussed.     

Bacillus subtilis deoxyribonucleic acid gyrase

Bacillus subtilis 168 was shown to contain a deoxyribonucleic acid (DNA) gyrase activity which closely resembled those of the enzymes isolated from Escherichia coli and Micrococcus luteus in its enzymatic requirements, substrate specificity, and sensitivity to several antibiotics. The enzyme was purified from the wild type and nalidixic acid-resistant and novobiocin-resistant mutants of B. subtilis and was functionally characterized in vitro. The genetic loci nalA and novA but not novB were shown to code for portions of the functional gyrase. Enzyme from the antibiotic-resistant mutants was resistant to the drug in vitro. The most striking observation was the remarkable similarity between the B. subtilis enzyme and other DNA gyrases, especially with respect to the oxolinic acid-induced DNA cleavage in the presence of sodium dodecyl sulfate. All of the enzymes appeared to possess the same specificity of cutting sites regardless of the source or type of DNA used. This result implies that gyrase binding to DNA is highly specific.     

Assimilation of single-stranded donor deoxyribonucleic acid fragments by nucleoids of competent cultures of Bacillus subtilis.

Lysates containing folded chromosomes of competent Bacillus subtilis were prepared. The chromosomes were supercoiled, as indicated by the biphasic response of their sedimentation rates to increasing concentrations of ethidium bromide. Limited incubation of the lysates with increasing concentrations of ribonucleases resulted in a gradual decrease in the sedimentation velocity of the deoxyribonucleic acid (DNA) until finally a constant S value was reached. Incubation with sonicated, 4,5',8-trimethylpsoralen-monoadducted, denatured, homologous donor DNA molecules at 37 degrees C and concomitant irradiation with long-wave ultraviolet light of the nucleoid-containing lysates resulted in the formation of complexes of the donor DNA molecules and the recipient chromosomes. This complex formation was stimulated when nucleoids were previously (i) unfolded by ribonuclease incubation, (ii) (partially) relaxed by X irradiation, or (iii) subjected to both treatments. Monoadducts were not essential. On the other hand, the complex-forming capacity of recipient chromosomes previously cross-linked by 4,5',8-trimethylpsoralen diadducts was greatly reduced, suggesting that strand separation of the recipient molecule was involved in the formation of the complex. None of these effects has been observed when heterologous (Escherichia coli) donor DNA has been used. When the same kind of experiments were carried out at 70 degrees C, donor-recipient DNA complexes were also formed and required strand separation and homology similar to donor-recipient complex formation at 37 degrees C. However, in contrast to what was found at 37 degrees C, unfolding plus relaxation of the nucleoids, as well as the absence of monoadducts in the donor DNA fragments, resulted in a decrease in complex formation. On the basis of these results, we assume that superhelicity can promote the in vitro assimilation of single-stranded donor DNA fragments by nucleoids of competents B. subtilis cells at 70 degrees C, but that at 37 degrees C a different mechanism is involved.     

Interactions between exogenous deoxyribonucleic acid and membrane vesicles isolated from competent and noncompetent Bacillus subtilis.

Competent cultures of Bacillus subtilis 168 have been fractionated into a high-competent and a low-competent fraction by a large-scale separation technique. Membrane vesicles isolated from both cell fractions are equally active in the transport of L-glutamate. Both membrane vesicle preparations seem to have similar endo- and exonuclease activities. Also, both preparations are capable of binding deoxyribonucleic acid. However, especially at low deoxyribonucleic acid concentrations (1 mug or less per ml), vesicles obtained from competent cells bind significantly more deoxyribonucleic acid (up to sixfold) than vesicles from noncompetent cells.     

Fate of heterologous deoxyribonucleic acid in Bacillus subtilis.

CsCl density gradient fractionation of cell lysates was employed to follow the fate of Escherichia coli, phage T6, and non-glucosylated phage T6 deoxyribonucleic acid (DNA) after uptake by competent cells of Bacillus subtilis 168 thy minus trp minus. Shortly after uptake, most of the radioactive Escherichia coli or non-glucosylated T6 DNA was found in the denatured form; the remainder of the label was associated with recipient DNA. Incubation of the cells after DNA uptake led to the disappearance of denatured donor DNA and to an increase in the amount of donor label associated with recipient DNA. These findings are analogous to those previously reported with homologous DNA. By contrast, T6 DNA, which is poorly taken up, appeared in the native form shortly after uptake and was degraded on subsequent incubation. The nature of the heterologous DNA fragments associated with recipient DNA was investigated with Escherichia coli 2-H and 3-H-labeled DNA. Association of radioactivity with recipient DNA decreased to one-fourth in the presence of excess thymidine; residual radioactivity could not be separated from recipient DNA by shearing (sonic oscillation) and/or denaturation, but was reduced by one-half in the presence of a DNA replication inhibitor. Residual radioactivity associated with donor DNA under these conditions was about 5% of that originally taken up. Excess thymidine, but not the DNA replication inhibitor, also decreased association of homologous DNA label with recipient DNA; but, even in the presence of both of these, the decrease amounted to only 60%. It is concluded that most, or all, of the Escherichia coli DNA label taken up is associated with recipient DNA in the form of mononucleotides via DNA replication.     

Interactions of homologous and heterologous deoxyribonucleic acids and competent Bacillus subtilis cells.

Glucosylated and nonglucosylated bacteriophage T4 deoxyribonucleic acids (DNAs) are able to bind to competent cells of Bacillus subtilis, although the former does so in a rather unstable fashion, probably because of the glucosylation. Several heterologous DNAs compete with homologous DNA for the same receptors in binding and in transformation. A different pattern in competition for DNA binding was observed for homologous and T4 glucosylated DNAs in intact cells as compared with protoplasts or membrane vesicles. The results are consistent with the existence of two types of receptor sites on the membrane of competent B. subtilis cells.     

Effects of chloramine on Bacillus subtilis deoxyribonucleic acid.

The lesions induced in Bacillus subtilis deoxyribonucleic acid (DNA) after treating bacterial cells (in vivo) and bacterial DNA (in vitro) with chloramine were studied biologically and physically. Single-strand breaks and a few double-strand scissions (at higher chloramine doses) accompanied loss of DNA-transforming activity in both kinds of treatments. Chloramine was about three times more efficient in vitro than in vivo in inducing DNA single-strand breaks. DNA was slowly chlorinated; the subsequent efficiency of producing DNA breaks was high. Chlorination of cells also reduced activity of endonucleases in cells; however, chlorinated DNA of both treatments was sensitized to cleavage by endonucleases. The procedure of extracting DNA from cells treated with chloramine induced further DNA degradation. Both treatments introduced a small fraction of alkali-sensitive lesions in DNA. DNA chlorinated in vitro showed further reduction in transforming activity as well as further degradation after incubation at 50 C for 5 h whereas DNA extracted from chloramine-treated cells did not show such a heat sensitivity.     

Single-stranded fraction of deoxyribonucleic acid from Bacillus subtilis.

About 13% of the deoxyribonucleic acid (DNA) of various strains of Bacillus subtilis, independent of the stage of growth or competence for transformation, was rendered acid soluble by endonuclease S1. In a pH 11.2 CsCl gradient, 4% of the untreated DNA banded at the density typical for single-stranded molecules, whereas 9% of the remaining DNA (main band) was sensitive to endonuclease S1. Selective inhibition of DNA polymerase III, or of DNA-dependent ribonucleic acid polymerase, did not increase or abolish single-strandedness. The DNA purification procedure did affect the level of single-stranded DNA, indicating its binding to cell constituents containing ribonucleic acid, protein, and membranous material. The molecular weight of the single-stranded fraction resembled that of total denatured DNA, and its buoyant density in an alkaline CsCl gradient was centered partially at a density of 1.772 g/cm3 and partially at a density of 7.759 g/cm3. Incubation of DNA under conditions leading to renaturation of its single-stranded fraction led to an increase in transforming activity for the purA16+ marker (close to the origin of replication) relative to leu-8+ and metC3+ markers (located in the middle of the chromosome), indicating this region is the main source of the single-stranded fraction.     

Changes in deoxyribonucleic acid polymerase activities in synthesis of deoxyribonucleic acid during sporulation of Bacillus subtilis.

The deoxyribonucleic acid (DNA) polymerase activities in Bacillus subtilis strains Marburg 168 (thy-trp2) and D22, a DNA polymerase I-deficient mutant, were measured at various stages of sporulation. The DNA polymerase I activity, which had decreased after the exponential growth, began to increase at the early stage of sporulation, reached a maximum and then again decreased. The activity of neither DNA polymerase II nor III was observed to change so drastically as that of DNA polymerase I during sporulation. The incorporation of [3H]deoxythymidine 5'-triphosphate ([3H]dTTP) into Brij 58-treated permeable cells increased during sporulation. The stimulation of [3H]dTTP incorporation into the cells by irradiation with ultraviolet light was also observed to coincide with DNA polymerase I activity. In strain D22 the activities of DNA polymerase II and III were almost constant with time. Neither change of [3H]dTTP incorporation into Brij 58-treated cells nor stimulation of incorporation by irradiation with ultraviolet light was observed.     

Formation of competent Bacillus subtilis cells.

The process of competent cell formation for transformation has been studied with early-stationary-phase (T1) cells of Bacillus subtilis which had been grown in an enriched Spizizen minimal medium and transferred to a second synthetic medium. Rifampin, chloramphenicol, and tunicamycin were strong inhibitors of competent cell formation, as well as vegetative growth. After formation, competent cells were no longer sensitive to the above agents. Methicillin and an inhibitor of chromosomal replication, hydroxyphenylazouracil, did not inhibit the development of competence. A D-alanine-requiring mutant strain developed competence even in the absence of D-alanine in the second medium. A T1-stage culture showed the activity of extracellular serine protease which is necessary for sporulation. Competent cell formation was completely blocked by 0.7 M ethanol, which is a specific inhibitor of early events during sporulation, including forespore septum formation. Competent cells were formed even in media which supported sporulation. The development of competence was also studied with spo0 mutants at 10 different loci. Most spo0 mutations repressed the development of competence except for spo0C, spo0G, and spo0J. These results suggest that competent cells are formed from early sporulating cells with the synthesis of cell wall materials and by factors whose genes are activated by the supply of nutrients. It is suggested that common steps are involved both in forespore septation and in competent cell formation.     

Fate of transforming deoxyribonucleic acid after uptake by competent Bacillus subtilis: phenotypic characterization of radiation-sensitive recombination-deficient mutants

A collection of 16 isogenic recombination-deficient strains of Bacillus subtilis isolated on the basis of sensitivity to methyl methane sulfonate (MMS) or mitomycin C (MC) were characterized phenotypically. All were found to be somewhat sensitive to ultraviolet irradiation, MC, and MMS. The mutants were all blocked in "late" steps in the transformation process and were provisionally grouped into four categories on the basis of the various properties examined. Class I mutants were deficient in transformation and heterologous transduction with phage PBS1 but were transducible with homologous donors at nearly the wild-type frequency. They were blocked in donor-recipient complex (DRC) formation but formed essentially normal amounts of double-strand fragments (DSF) and single-strand fragments (SSF). The class IIa strain was deficient in transformation and PBS1 transduction, and formed DRC which was normal by all available physical and biological criteria. Class IIb mutants were deficient in transformation and PBS1 transduction, and failed to form DRC. They did produce DSF and SSF. Class III mutants were deficient in transformation, were normal in PBS1 transduction, and formed DRC which was physically indistinguishable from that of the Rec(+) parent although with slightly lowered donor-type transforming activity. Class IV strains were deficient in PBS1 transduction but were transformed at nearly the wild-type efficiency. None of the mutant strains was deficient in the adenosine triphosphate-dependent deoxyribonuclease.     

Transformation of a Bacillus subtilis L-form with bacteriophage deoxyribonucleic acid.

A stable L-form, sal-1, of Bacillus subtilis was transformed with deoxyribonucleic acid (DNA) from bacteriophages phi 25 and phi 29 to determine whether exogenous DNA can be introduced into this organism. The viral transformation (transfection) was successful with the use of polyethylene glycol. In the presence of the fusogen, bacteriophage phi 25 DNA initiated a single cycle of infection. When compared with transfection of competent cells of Bacillus subtilis, the appearance of viral particles was delayed and their production occurred over a longer time period. L-form cells were best able to support intracellular replication of phi 25 viral particles when in balanced growth in a rich medium. The addition of polyethylene glycol also induced infection of sal-1 with whole bacteriophage phi 25 particles which could not otherwise infect the L-form and enhanced infection by intact phi 29 particles. Primary recombination was shown to be required for polyethylene glycol-mediated phi 25 transfection, but not phi 29 transfection or for whole bacteriophage phi 25 infection mediated by polyethylene glycol. Successful transfection of sal-1 suggests that the L-form may be amenable to genetic modification with exogenous DNA.     

Identification of a sporulation locus in cloned Bacillus subtilis deoxyribonucleic acid.

A cloned deoxyribonucleic acid from the purA-cysA region of the Bacillus subtilis chromosome was shown to contain the spoVC locus, a gene whose product is required for sporulation. This is the first demonstration of a spo locus in cloned B. subtilis deoxyribonucleic acid.     

DNA synthesis in competent Bacillus subtilis cells.

Competent cells of Bacillus subtilis incorporate degradation products from transfecting DNA into their chromosomal DNA. The sensitivity of this incorporation to inhibitors of bacterial DNA synthesis [phage infection or 6-(p-hydroxyphenylazo)-uracil] suggests that semiconservative DNA synthesis can occur in competent cells.     

Some properties of a membrane-deoxyribonucleic acid complex isolated from Bacillus subtilis.

Membrane-deoxyribonucleic acid (DNA) complexes were isolated from Bacillus subtilis by affinity for magnesium-Sarkosyl crystals. These complexes (M-bands) contained greater than 80% of the total cellular DNA; little of the remaining portion could be recovered in a secondary isolation. Isotopic labeling of the origin of replication showed this region of the chromosome to be closely associated with the cell membrane. Interruption of protein or DNA synthesis did not result in detachment of the chromosome from the membrane. Interruption of ribonucleic acid synthesis by rifampin resulted in a decreased ability to isolate DNA in the M-band. Analysis of attachment of the chromosome to membrane during the cell and replication cycles indicated that the chromosome is not released from the membrane at any time during the cell cycle.     

Temperature-sensitive deoxyribonucleic acid replication in a dnaC mutant of Bacillus subtilis.

A temperature-sensitive mutant of Bacillus subtilis is defective in deoxyribonucleic acid (DNA) synthesis, contains a lesion in the dnaC locus, and is not primarily an initiation mutant. The amount of DNA synthesized by this mutant at temperatures above 40 C decreases with increasing temperature. DNA synthesis resumes within 20 min after the temperature is lowered to 30 C. In the presence of chloramphenical, DNA synthesis begins at a reduced rate after the temperature is lowered to 30 C. Spores germinated at 46 C cannot initiate DNA replication. The capacity for residual DNA synthesis is stable at the restrictive temperature during inhibition of DNA synthesis. When the temperature is lowered to 30 C after a period of incubation at 43 C, DNA synthesis starts at the origin of the chromosome as well as at preexisting growing points. Similar DNA synthesis patterns are found in mutant cells in vivo and after toluene treatment.