Fractionation of DNA from Bacillus subtilis and its transforming activity for various markers

The physical, chemical, and functional heterogeneity of tranforming DNA was studied by preparative fractionation techniques providing resolution with respect to differences in molecular weight (gel filtration, sucrose density gradient), base composition (CsCl density gradient), or both these parameters simultaneously (methylalbumin-coated celite 545 MAK column). A comparison of the basic characteristics of the obtained fractions (melting temperature, T _m; density, ; sedimentation coefficient, S _20,w; and transforming activity for ade, leu, and met markers) showed that the factor decisive for functional activity represents, in addition to the sequential arrangement of nucleotides in the chain, the average base composition. Hence, using the methylalbumin column or CsCl density gradient centrifugation, DNA fractions can be isolated which show a several times higher transforming activity for any of the markers examined. By contrast, the remaining fractionation methods, even though considerably decreasing the heterogeneity of the fractions as regards their molecular weight (such as zonal centrifugation), do not offer a possibility of fractionation of the activity for individual markers. This indicates a statistically random degradation of transforming DNA during its isolation. The order of the investigated markers according to their guanine-cytosine content is ade leu met and corresponds also to the order of their positions on the genetic map of Bacillus subtilis.     

Mapping by interspecies transformation experiments of several ribosomal protein genes near the replication origin of Bacillus subtilis chromosome.

Summary Bacillus subtilis 168 was transformed with DNAs from B. amyloliquefaciens K or B. licheniformis IAM 11054. These two species show a considerable difference in ribosomal proteins from B. subtilis. Analyses of the transformants indicated that the genes for 16 proteins, S3, S5, S8, S12, S17, S19, BL4, BL5, BL6, BL8, BL14, BL16, BL17, BL22, BL23 and BL25 are located in the cysA-str-spc region on B. subtilis chromosome. The genes for 10 proteins, S4, S6, S13, S16, S20, BL15, BL18, BL20, BL24 and BL28 could not be found in this region in the prsent experiments.     

Membrane enrichment of genetic markers close to the origin and terminus during the deoxyribonucleic acid replication cycle in Bacillus subtilis.

A temperature-sensitive Bacillus subtilis initiation mutant was used to achieve one cycle of synchronized deoxyribonucleic acid (DNA) replication. Markers near the origin of replication and the terminus were assayed for association with the cell membrane at intervals during the DNA replication cycle. DNA near the origin and terminus was found to be enriched in the membrane fraction throughout the DNA replication cycle. The magnitude of membrane enrichment or origin and terminus markers varied coincidentally, possibly as a consequence of incubating the cells at 45 degrees C.     

A protein involved in termination of chromosome replication in Bacillus subtilis binds specifically to the terC site.

The small basic protein encoded by the open reading frame adjacent to the terC site in the Bacillus subtilis chromosome and previously implicated in termination of the replication process was purified. Band retardation assays established that this protein (now called the replication terminator protein, encoded by the rtp gene) binds specifically to a 209-base-pair fragment of DNA within which terC is located.     

Localization of a DNA replication origin and termination zone on chromosome III of Saccharomyces cerevisiae.

Two-dimensional gel electrophoretic replicon mapping techniques were used to identify all functional DNA replication origins and termini in a 26.5-kbp stretch in the left arm of yeast chromosome III. Only one origin was detected; it coincided with an ARS element (ARS306), as have all previously mapped yeast origins. A replication termination region was identified in a 4.3-kbp stretch at the telomere-proximal end of the investigated region, between the origin identified in this paper and the neighboring, previously mapped, ARS305-associated origin (previously called the A6C origin). Termination does not occur at a specific site; instead, it appears to be the consequence of replication forks converging in a stretch of DNA of at least 4.3 kbp.     

Sequence limits of DNA strands in the arrest replication fork at the Bacillus subtilis chromosome terminus.

The DNA sequence limits of the leading and lagging strands in the arrested clockwise replication fork at the terminus of the Bacillus subtilis chromosome have been investigated. On the basis of hybridization to synthetic oligonucleotides corresponding to known positions in the terminus region sequence it has been shown that neither the leading nor lagging strands, as they approach terC, traverse the distal inverted repeat, IRI. But a small fraction of the leading strands pass through the proximal inverted repeat, IRII. This is consistent with IRI being the functional inverted repeat in arresting the clockwise fork. But most of the forks appear to stop at least 100 nucleotides short of IRI, and at various positions extending over a distance of at least 100 nucleotides.     

Regulation of initiation of the chromosomal replication by DnaA-boxes in the origin region of the Bacillus subtilis chromosome.

A gene homologous to the Escherichia coli dnaA gene and two flanking 'regulatory' regions which contain nine and four DnaA-boxes respectively, are located in the replication origin region of the Bacillus subtilis chromosome. Attempts to isolate an autonomously replicating fragment from these 'regulatory' regions in order to identify oriC have been unsuccessful because the DnaA-box-containing regions strongly inhibited plasmid transformation particularly when inserted into a high-copy number plasmid pUB110. Using two plasmids differing in copy number, the two regions were subdivided into three regions, A, B and C, each containing five, four and four DnaA-boxes respectively, which differed in level of inhibition of transformation. Region C is downstream of the 'dnaA' gene and inhibits transformation in high-copy but not in low-copy number plasmids. When a part of the DnaA-boxes was deleted from the incompatible plasmids, they became transformable and produced slow-growing transformants in which the initiation frequency of chromosomal replication was selectively reduced. Fast-growing revertants were found containing the same number of plasmids as the parent but with single base changes in the DnaA-boxes. These mutations were in the most highly conserved bases of the DnaA-box sequence. This indicates that a sequence-specific interaction of the DnaA-box, probably with the B. subtilis DnaA protein is responsible for the observed incompatibility and thus appears to be involved in control of initiation frequency of the chromosomal replication.     

DNA of Bacillus subtilis bacteriophage SPP1: physical mapping and localization of the origin of replication.

The genome of Bacillus subtilis bacteriophage SPP1, a linear, 28.5-megadalton DNA duplex, was mapped by analysis with the restriction endonucleases endo R.Sal I, Sma I, Xba I, Bgl I, Bgl II, and EcoRI. The SPP1 genome, like that of the Salmonella typhimurium phage, P22, was found to be a terminally repetitious, circularly permuted molecule. 6-(p-Hydroxyphenylazo)uracil, a selective, reversible inhibitor of SPP1 DNA synthesis, was exploited to synchronize the initiation of genome replication and to selectively label the site of its initiation with radioactive thymidine. Restriction endonuclease analysis of the distribution of the label located the origin of replicative synthesis at an area approximately 0.2 genome length from one molecular terminus.     

Operon of riboflavin biosynthesis in Bacillus subtilis. XVI. Localization of the ribC-group markers on the chromosome]

Regulatory markers of ribC group were located on the chromosome of Bacillus subtilis by means of genetic transformation. Markers of this group controlling the regulation of riboflavin biosynthesis were mapped between markers of resistance to acriflavin and streptomycin (strC group). The value of cotransfer index between acriflavin-resistance markers and ribC markers was found to be 26--32%. Acriflavin inhibits the riboflavin biosynthesis. The level of inhibition depends on the genotype of riboflavin-producing strains, while the inhibition of the cell growth does not depend on it.     

Isolation of a DNA-protein complex containing a single DNA fragment which is at or near the replication origin of the Bacillus subtilis chromosome

A chromosomal fragment containing purA, the nearest marker from the replication origin of the Bacillus subtilis chromosome, was highly purified as a complex containing at least proteins and being solubilized easily during cell lysis. The complex had a markedly higher sedimentation rate (70–120S) than the bulk of the solubilized DNA (40S). The electron microscopic observation showed the complex to be an aggregate of several DNA molecules with a local structure containing amorphous materials which stained black and bushes of RNA. This confirmed biochemical evidences suggesting that the complex is an intermolecular aggregate of the purA-DNA-protein-RNA complex.     

Functional evidence that the principal DNA replication origin of the Streptomyces coelicolor chromosome is close to the dnaA-gyrB region.

The degree of overrepresentation of selected chromosomal genes in rapidly growing cultures of Streptomyces coelicolor was assessed by quantitative DNA hybridization analysis. The results are consistent with the hypothesis that the principal origin of replication is close to the dnaA-gyrB region, in the center of the linear chromosome, and that replication proceeds bidirectionally.     

Fate of transforming DNA following uptake by competent Bacillus subtilis. I. Formation and properties of the donor-recipient complex

Following uptake by competent Bacillus subtilis, transforming DNA is converted to two distinct slowly sedimenting molecular forms which possess little transforming activity (eclipse). A few minutes after uptake is initiated, a physical complex of donor and recipient DNA begins to form. The recovery of donor transforming activity following eclipse, and the appearance of recombinant activity, previously reported by Venema, Pritchard &; Venema-Schröder (1965), is shown to be due to changes occurring in the donor—recipient complex. This complex exists transiently in a form with low recombinant-type transforming activity. This transient form may be one in which the donor and recipient components are joined non-covalently. The donor-recipient complex is shown to be a heteroduplex structure in which the donor moiety has an approximate molecular weight of 750,000.     

Heterologous transformation in Bacillus subtilis. 1. Transmission of DNA of the R1drd19 plasmid]

Bac. subtilis 168 (BD-25) cells were infected with DNA of plasmide R1drd19 isolated from E. coli strain; transformants resistant to streptomycin (500 microgram/ml) and kanamycin (40 microgram/ml) appeared with the frequency of 2.10(-6). These transformants retained resistance to the mentioned antibiotics stably. A satellite DNA peak was revealed in centrifugation in the density gradient of cesium chloride with ethidium bromide. It was possible to infect cells of Bac. subtilis 168 (BD-25) with plasmide DNA isolated from the transformants. Plasmide transduction with the aid of phages AR9 and PBSI multiplied on the transformant strains was also effected. Physico-chemical analysis of the transformed plasmide DNA was conducted; its molecular weight was determined.     

Initiation and termination of chromosome replication at 45 degree C in a temperature-sensitive deoxyribonucleic acid initiation mutant of Bacillus subtilis 168, TsB134.

Deoxyribose nucleic acid transfer experiments showed that upon shifting Bacillus subtilis TsB134 to 45 degree C, initiation of new rounds of replication was effectively blocked and the majority of existing rounds terminated.     

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. III. Nucleotide sequence of some 10,000 base pairs in the origin region.

Approximately 10,000 nucleotides were sequenced in the oriC region of the Bacillus subtilis chromosome. The first replicating DNA strands are hybridized with a SalI-EcoRI fragment (nucleotide #1206-2954) in one direction (left to right) and an EcoRI-PstI fragment (#2949-4233) in the other. Seven open reading frames (ORF) accompanied with Shine-Dalgarno (SD) sequences were identified. ORF638 and ORF821 were identified as gyrB and gyrA genes respectively based on genetic evidences and amino acid sequence data. Comparison of amino acid sequences revealed that ORF44, ORF446, ORF378 and ORF323 are homologous with rpmH, dnaA, dnaN and recF of Escherichia coli, respectively. Thus, the organization of the ORFs from ORF44 to ORF638 resembles the organization of genes in the rpmH-gyrB region of the E. coli chromosome. Two non-coding regions characteristic for oriC signals were found near the site of initiation of the first replicating DNA. They are composed of repeating sequences whose consensus sequence TTAT(C/A)CACA is identical to that of 4 repeating sequences in the oriC of E. coli.