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.     

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.     

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.     

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.     

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.     

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.     

Mutation induction by the direct treatment of Bacillus subtilis deoxyribonucleic acid.

A procedure was developed to select for specific mutations obtained by means of transformation with DNA previously exposed to potentially dangerous chemical compounds. The 70% co-transformation of hisB and trpC genes in Bacillus subtilis provided a convenient opportunity to select for new mutations. When purified DNA from wild type bacteria was treated with N(OH) acetyl aminofluorene or Hoechst dye 37 507 and used to transform a recipient bearing of a trpC2 mutaion, a high proportion of the Trp+ transformants had new hisB mutations.     

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.     

Postreplication repair of ultraviolet-irradiated transforming deoxyribonucleic acid in Bacillus subtilis

Repair of ultraviolet-irradiated transforming deoxyriboinucleic acid (DNA) in several strains of Bacillus subtilis was studied in order to determine the effects of excision repair and postreplication repair on transformation. Two mutations that cause a Uvr- and phenotype (uvr-1 and uvr-42) were shown to have strikingly different effects on repair of ultraviolet-irradiated transforming DNA. Genetic and kinetic evidence is presented to show that integrated DNA was apparently repaired by both excision and postreplication repair in wild-type and in uvr-1 recipients, although the latter excise pyrimidine dimers very slowly. In uvr-42 mutants, which are defective in incision at pyrimidine dimers, dimer-containing DNA was integrated. Postreplication repair apparently saved uvr-42 recipient cells from the lethal effects of integrated dimers, but the recombination events accompanying postreplication repair greatly reduced the linkage between closely linked genetic markers in the donor DNA. Repair of transforming DNA in a recG recipient, which does excision repair but not postreplication repair, was nearly as efficient as in wild-type cells. However, in this recipient linkage was altered only slightly, if at all, compared with wild-type cells. The apparent reduction in size of integrated regions of ultraviolet-irradiation transforming DNA probably results mainly from postreplication repair of larger integrated regions.     

Genetic transformation with cell wall-associated deoxyribonucleic acid in Bacillus subtilis

Cell walls from bacillus subtilis 168 were prepared by conventional methods and found to contain deoxyribonucleic acid (DNA). In transformation assays, after autolysis, it was found that two major regions of the chromosome were selectively enriched in the wall preparations. One region clustered around the replication origin and is represented by the markers purA16, ts8132, thiC5, sacA321, and hisA1. The other region included the replication terminus with representative loci metB10, citK5, gltA292, and pyrA1. All other (internal) loci which were examined showed no statistical enrichment. The two areas of enrichment were similar to but more extensive than those reported for membrane-DNA complexes. The wall preparations also contained protein and lipid, indicating a possible membrane involvement. Analyses of the cell walls revealed that the fatty acid composition of the membrane component was not typical of the for B. subtilis protoplast membranes or for lipoteichoic acids. In addition, radioiodination of cell wall autolysates, followed by gel electrophoresis and autoradiography, demonstrated the presence of proteins not readily detectable in bulk protoplast membranes or on the surfaces of intact cells. These data suggest that a unique component of the membrane and regions of the B. subtilis genome involved in DNA replication events are tightly associated with cell walls. The binding of DNA-membrane complexes to the "rigid" cell wall and the replication of the wall could be a mechanism by which the segregation of growing chromosomes occurs.     

Cell wall synthesis and initiation of deoxyribonucleic acid replication in Bacillus subtilis.

We have observed a connection between cell wall synthesis and the initiation of chromosome replication in Bacillus subtilis. Initiation of chromosome replication was prevented in synchronous cultures in the presence of the cell wall synthesis inhibitor vancomycin. When vancomycin was added to the cultures after initiation of chromosome replication, one round of replication was completed but no reinitiation occurred. Similar results were obtained when cell wall synthesis was inhibited by ristocetin, cycloserine, cloxacillin, or cephaloridine. When sucrose was added to the medium, initiation of deoxyribonucleic acid replication occurred in the presence of vancomycin, to an extent which allowed replication of no more than approximately one-half of the deoxyribonucleic acid of the culture. The same was found in cultures of spheroplasts of B. subtilis. However, initiation of chromosome replication in spheroplasts was completely insensitive to cloxacillin.     

Repair and subsequent fragmentation of deoxyribonucleic acid in ultraviolet-irradiated Bacillus subtilis recA.

Cells of Bacillus subtilis recA1 are sensitive to irradiation with ultraviolet light. Evidence is presented here that these cells are not defective in ultraviolet light-induced incision of deoxyribonucleic acid (DNA) or repair DNA synthesis. Ligation of DNA at repair sites appears to occur, but the DNA is subsequently fragmented, apparently at sites of previous repair synthesis. It is hypothesized that the defect in DNA repair leads to host-specific restriction at repaired sites because of a defect in either the structure of the repaired region or specificity of the restriction/modification system.     

Involvement of deoxyribonucleic acid polymerase III in W-reactivation in Bacillus subtilis

6-(p-Hydroxyphenylazo)-uracil, a purine analog that is known to specifically inhibit deoxyribonucleic acid polymerase III in gram-positive organisms, inhibited W-reactivation in Bacillus subtilis. On the other hand, W-reactivation proceeded normally in the presence of 6-(p-hydroxyphenylazo)-uracil when a strain possessing a resistant deoxyribonucleic acid polymerase III was used as the host.     

Fractionation of native and denatured transforming deoxyribonucleic acid from Bacillus subtilis

1. Native DNA from Bacillus subtilis was fractionated by stepwise elution from methylated albumin, the transforming activity being confined to two out of four fractions. Partial separation of DNA active in transformation for the arginine marker from that showing activity for the histidine and tryptophan markers was achieved. 2. Partial denaturation of DNA at 90 degrees and 93.5 degrees resulted in the preferential destruction of transforming activity for the histidine and tryptophan markers. 3. Denaturation of DNA at 100 degrees followed by chromatography on methylated albumin yielded five fractions, two of which exhibited residual activity. Redenaturation at 100 degrees resulted in the interconversion of four out of the five fractions. Redenaturation of fractions labelled with (15)N and (2)H suggested the presence of a specific component that did not readily take part in the interconversions.