Possible involvement of DNA-linked RNA in the initiation of Bacillus subtilis chromosome replication.

After thermal denaturation, an in vivo-labeled RNA was found in a temperature-sensitive initiation mutant of Bacillus subtilis (dna-37) associated with high-molecular-weight DNA. This RNA could be clearly distinguished from other RNA species by different techniques of separation, such as Sepharose 2B filtration, chromatography on nitrocellulose, and equilibrium centrifugation in density gradient. It was obtained even when HCHO was present during denaturation and chilling of nucleic acids and was still detected after a second denaturation as well as after incubation with proteinase K. Properties of the complex were not altered by prior treatment with RNase H. A control experiment using two samples of the complex treated either with pancreatic DNase or with pancreatic RNase, denatured together and centrifuged in the same density gradient, showed that no artifactual associations occur between the DNA and the RNA components of the complex. These results demonstrate that the DNA and RNA in the complex are associated by neither hydrogen bonds nor proteins, but are indicative of a DNA-RNA covalent linkage. In addition, during synchronous replication after a previous period at a nonpermissive temperature, DNA-linked RNA synthesis took place at specific times which coincided with the appearance of rifampin resistance of the first and the second replication cycles. A possible involvement of this RNA in the initiation of chromosome replication is discussed.     

Sporulation in Bacillus subtilis. Effect of medium on the form of chromosome replication and on initiation to sporulation in Bacillus subtilis

Thymine-requiring mutants of Bacillus subtilis and mutants that are temperature-sensitive for initiation of chromosome replication have been used to study the relationship between sporulation and chromosome formation. The DNA synthesis that normally occurs when cells are transferred to sporulation medium is essential for spore induction. This is shown by the fact that thymine-starved cells are unable to form spores and are unable to perform even the earlier steps of sporulation, such as septum formation or synthesis of alkaline phosphatase. The nature of the medium in which the cells are growing while the DNA is being completed is also important because it determines both the shape and the position of the daughter chromosomes. If the cells are in a rich medium, the newly synthesized chromosomes are discrete and compact bodies: the cells are primed for growth, and sporulation cannot be induced by transferring them at this stage to a spore-inducing medium. If DNA synthesis was completed with the cells in a poor medium the daughter chromosomes, by the time DNA synthesis has ceased, are spread in a single filamentous band and the cells are morphologically already in stage I of sporulation.     

Bidirectional chromosome replication in Bacillus subtilis 168.

Density transfer analysis of deoxyribonucleic acid from Bacillus subtilis 168 thy spores germinating in 5-bromouracil medium shows the order of replication of genetic markers to be: purA16, cysA14, sacA, ctrA, (narB, arol), dal, (hisA1, purB6), (tre-12, thr-5), (argA, aroG, argC4), (metC, leu-8, pheA), (ura-1, aroD), lys-1, (trpC, metB, ilvA, citB, citK, gltA). The precise order of transfer of markers within parentheses could not be determined in these experiments. Taken together with new PBS1 transduction data presented here and in the accompanying paper of J. Lepesant-Kejzlarová, J.-A. Lepesant, J. Walle, A. Billaut, and R. Dedonder (1975), the results can be resolved in terms of a symmetric, fully bidirectional mode of chromosome replication with a replication origin close to the purA16 marker and a terminus in the region of the gltA, citK loci, diametrically opposed to the origin. A new genetic map of the B. subtilis 168 chromosome is presented.     

Control of initiation of sporulation by replication initiation genes in Bacillus subtilis

Initiation of spore formation in Bacillus subtilis appears to depend on initiation of DNA replication. This regulation was first identified using a temperature-sensitive mutation in dnaB. We found that mutations in the replication initiation genes dnaA and dnaD also inhibit sporulation, indicating that inhibition of sporulation is triggered by general defects in the function of replication initiation proteins.     

Macromolecular synthesis in chromosome initiation mutants of Bacillus subtilis

Molecular Genetics and Genomics - Inactivation of the dna B or dna D gene product in Bacillus subtilis stimulates RNA and protein synthesis. Strains containing ts dna B and D mutations have been...     

A new relaxed mutant of Bacillus subtilis.

A new relaxed mutant of Bacillus subtilis was isolated by screening Rifr clones for alterations in stringent control. The Rifr relaxed mutant which is described was found to contain a second-site mutation conferring a relaxed response to an energy source downshift and was partially relaxed after amino acid starvation. The new rel locus, called relG, was distinct from the two other known rel loci in B. subtilis, relA, and relC.     

Anucleate cell production and surface extension in a temperature-sensitive chromosome initiation mutant of Bacillus subtilis.

At 45 C, in a temperature-sensitive initiation mutant (TsB134) of Bacillus subtilis 168 Thy- tryp-, growing in a glucose-arginine minimal medium, chromosome completion occurred over a period of 80 to 90 min, after which there was no further nuclear division. Normal symmetrical cell divisions continued for a generation afterwards, so that nuclei were segregated into separate cells. During this period asymmetric divisions started to occur. Septa appeared at 25 to 30% from one end of the cell, giving a small anucleate cell and a larger nucleate cell. During inhibition of deoxyribonucleic acid (DNA) synthesis by thymine starvation under the restrictive conditions, asymmetrical division also occurred until there was approximately one nucleus per cell (about one generation time). Asymmetric division, giving anucleate cells, then occurred. Similar results were obtained when DNA synthesis was inhibited by nalidixic acid. After 3 h at 45 C, the rate of anucleate cell production in the presence and absence of thymine was constant at one division per 85 min per chromosome terminus present when DNA synthesis stopped. In the absence of DNA synthesis (during thymine starvation) at 35 C, growth in cell length was linear (i.e., the rate was constant), but at 45 C during thymine starvation the rate gradually increased by more than twofold. It is suggested that this was due to the establishment of new sites of growth associated with anucleate cell production. In the presence of thymine at 45 C, the rate of length extension increased by more than fourfold, which it is suggested was caused by the appearance of new growth zones as a result of chromosome termination and a contribution associated with anucleate cell production. If the mutant was incubated at 45 C for 90 min, both in the presence and absence of thymine, then anucleate cell formation could continue on restoration to 35 C in the absence of thymine...     

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.     

Macromolecular synthesis in chromosome initiation mutants of Bacillus subtilis.

Summary Inactivation of the dna B or dna D gene product in Bacillus subtilis stimulates RNA and protein synthesis. Strains containing ts dna B and D mutations have been constructed by introducing the mutations by transformation into a thymine requiring strain which does not lyse during thymine starvation. The consequences of inactivation of these gene products have been assessed by comparing RNA and protein synthesis during thymine starvation at the restrictive temperature with the recipient strain. In the ts ⁺ strain, there is a doubling in rate of RNA synthesis during thymine starvation. In the ts dna B and D mutations at the restrictive temperature the rate of RNA synthesis increases four fold. By preincubating the mutants in the absence of thymine for one generation at the permissive temperature the two fold increase in rate of RNA synthesis associated with inactivation of the initiation complex can be demonstrated under conditions where the ts ⁺ strain shows a decrease in rate of RNA synthesis. The rate of protein synthesis observed largely reflects the rate of RNA synthesis in all strains. Completion of the chromosome at the restictive temperature has no significant effect on the rate of RNA synthesis. It is suggested that inactivation of the initiation complex after chromosome initiation could play an important role in control of RNA synthesis in relation to the cell cycle.     

A temperature sensitive mutant in Bacillus subtilis with an altered elongation factor G

Summary A temperature sensitive mutant of Bacillus subtilis with an altered elongation factor G is described. The mutation is highly co-transformable with resistance to fusidic acid.This work is part of a Ph. D. Thesis to be submitted to Tel Aviv University.     

Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome

To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of ∼ 4 kb. The replicon was derived from a broad-host-range plasmid, pAMβ1, which replicates by a unidirectional theta mechanism and is thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and ∼ 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.     

Bacillus subtilis mutant with altered ability for heterologous transformation]

Mutant strain of Bacillus subtilis, which produced in certain conditions significantly reduced quantity of trnasformants during transformation by homologous DNA, as compared with transformation by heterologous DNA from Bac. aterrimus, is isolated. The ability to transfection by phage SPO1 DNA and the efficiency of infection of the mutant by this phage are also decreased. The causes of such alterated properties are discussed.     

Permanent loss of chromosome initiation in toluene-treated Bacillus subtilis cells.

Initiation of deoxyribonucleic acid replication is absent in Bacillus subtilis cells made permeable by toluene. The absence of initiation may be (i) a temporary removal of toluene, or (ii) irreversibly lost due to damage by toluene treatment to a cellular structure or a process required for chromosome initiation. Washed cells, previously treated with toluene and subsequently washed free of detectable amounts of toluene, have the same characteristics of toluene-treated cells in which toluene is not removed. The continued absence of initiation in the washed cells indicates a permanent loss of initiation in cells treated with toluene. Protein synthesis is also inhibited irreversibly by toluene treatment, indicating damage to translation as a possible mechanism for loss of initiation of toluene-treated cells.     

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.     

Termination of Bacillus subtilis chromosome replication as visualized by autoradiography

A mutant of Bacillus subtilis W23 thy his, temperature sensitive for the initiation of rounds of chromosome replication, has been used to investigate the manner in which the two growing points on the replicating circular chromosome approach one another during termination (completion) of the round in progress.The mutant, growing exponentially at 34 °C, was transferred to 45 °C and [³H]thymidine added shortly afterwards. After a further short interval, the specific radioactivity was lowered by a factor of three and rounds of replication were allowed to complete under these conditions. The pattern of heavy and light grain density regions in chromosomal structures made visible by autoradiography was examined, and many with both ends more heavily labelled than their internal region were found. From an analysis of the relative frequency and size of these structures it is concluded that the two growing points in a single chromosome can continue to move at similar rates (within a factor of 2) as they approach one another to within < 10% of the length of the whole circular chromosome in the vicinity of the terminus. The data obtained do not prove that all chromosomes behave in this manner, but are consistent with such an interpretation.     

Sequence features of the replication terminus of the Bacillus subtilis chromosome.

The sequence of 1267 nucleotides spanning the replication terminus, terC, of the Bacillus subtilis 168 chromosome has been determined. The site of arrest of the clockwise fork, which defines terC, has been localized to a 30-nucleotide portion (approximately) within this sequence. The arrest site occurs in an A + T-rich region between two open reading frames and very close to one of two imperfect inverted repeats (47-48 nucleotides each) which are separated by 59 nucleotides. The closeness of approach of the arrested clockwise fork to the first imperfect inverted repeat encountered in this region raises the possibility of a role for the inverted repeats in the mechanism of fork arrest.