DNA replication by a DNA-membrane complex extracted from Bacillus subtilis: site of initiation in vitro and initiation potential of subcomplexes.

A DNA-membrane complex extracted from Bacillus subtilis was studied further as a model system for initiation of bacterial DNA replication in vitro. Of three subcomplexes purified from the crude complex by a combination of CsCl and sucrose gradient centrifugation, the synthetic capability of only one was inhibited significantly by streptovaricin, a known inhibitor of RNA primer formation. A selective enrichment in the level of this subcomplex was obtained by manipulating a thymine-requiring mutant. The synthetic capabilities of an enriched and nonenriched DNA-membrane complex were compared in the presence and absence of streptovaricin. Although the rate and extent of DNA synthesis per unit of protein were approximately the same in the absence of the antibiotic, there was a much greater inhibition of synthesis shown by the enriched complex in the presence of streptovaricin. Although the amount of DNA present in the putative initiation subcomplex was less than 0.3 to 0.4% of the total DNA present in the crude complex, such DNA, except for a few quantitative differences, was still representative of genomic DNA. Newly synthesized DNA hybridized to specific origin- and non-origin-derived restriction fragments of the B. subtilis genome. However, when an elongation inhibitor (ddCTP) was added, hybridization of such DNA to almost all of the nonorigin fragments disappeared or was reduced drastically, whereas origin region hybridization patterns remained strong. The highest level of hybridization in the origin region occurred with a BamHI (B7) restriction fragment of 5.6 kilobases that has been implicated by others as one site initiation in vivo (N. Ogasawara, M. Seiki, and H. Yoshikawa, Nature (London) 281:702-704, 1979; S. J. Seror-Laurent and G. Henckes, Proc. Natl. Acad. Sci. USA 82:3586-3590, 1985).     

Oligonucleotide bias in Bacillus subtilis: general trends and taxonomic comparisons.

We present a general analysis of oligonucleotide usage in the complete genome of Bacillus subtilis . Several datasets were built in order to assign various biological contexts to the biased use of words and to reveal local asymmetries in word usage that may be coupled with replication, the control of gene expression and the restriction/modification system. This analysis was complemented by cross-comparisons with the complete genomes of Escherichia coli , Haemophilus influenzae and Methanococcus jannaschii . We have observed a large number of biased oligonucleotides for words of size up to 8, throughout the datasets and species, indicating that such long strict words play an important role as biological signals. We speculate that some of them are involved in interactions with DNA and/or RNA polymerases. An extensive analysis of palindrome abundances and distributions provides the surprising result that prophage-like elements embedded in the genome exhibit a smaller avoidance of restriction sites. This may reinforce a recently proposed hypothesis of a selfish gene phenomena in the transfer of restriction/modification systems in bacteria.     

Separation of two functional roles of L-alanine in the initiation of Bacillus subtilis spore germination

Spores of the standard transformable Marburg strain of Bacillus subtilis can be initiated to germinate by l-alanine alone. We isolated mutants which required for this process, in addition to l-alanine, the combination of d-glucose + d-fructose + K(+) or NH(4) (+) ions. In place of fructose, autoclaved or caramelized glucose could be used. Even the standard type strain required the addition of these three agents when d-alanine was present or when the temperature was raised. These findings show that l-alanine normally performs two functions during initiation, one of which is absent in the mutants or is blocked by d-alanine or elevated temperature. One of our mutants was not absolutely dependent on the addition of external l-alanine, because it could be initiated at a reduced rate by the sole addition of glucose + K(+) or NH(4) (+). When K(+) or NH(4) (+) was replaced by Na(+), the initiation rate was greatly reduced. The divalent metal ions Mg(++), Mn(++), and Ca(++) could not satisfy the cation requirement.     

Translationally coupled initiation of protein synthesis in Bacillus subtilis.

The neomycin phosphotransferase gene (neo) from Transposon Tn5 is active in Gram-negative bacteria but silent in B. subtilis since it lacks an appropriate ribosome binding site for Gram-positive bacteria. Neo translation could be reactivated by coupling its initiation to the translational termination of the highly expressed beta-lactamase gene (penP) from B. licheniformis. This initiation occurred at the authentic neo start codon. Its efficiency was independent of the nucleotide sequence 5 to the neo gene, but strongly affected by the distance between the termination and initiation codon. It was the highest if both codons overlapped in the sequence ATGA. In B. licheniformis, a translationally coupled neo gene was inducible expressed as the penP gene demonstrating the potential of the technique to monitor the activity of expression units for which no direct assays exists.     

Division septation in the absence of chromosome termination in Bacillus subtilis.

Germinating spores of the temperature-sensitive DNA initiation mutants of Bacillus subtilis, TsB134 and dna-1(Ts), were allowed to undergo a single round of replication by shifting to the restrictive temperature shortly after its initiation. To monitor the progress of the round 5-bromouracil was added at various times and DNA extracted after a further time, sufficient to allow completion of the chromosome. Average replication was measured from the relative amounts of LL and LH material in Cs₂SO₄ gradients. The replication state of origin (purA), intermediate (leuA) and terminus (metB) markers at the times of 5-bromouracil addition were obtained from genetic analysis of the density species fractionated in gradients of CsCl.The DNA replication inhibitor, 6-(p-hydroxyphenylazo)-uracil (HPUra), was added at various stages of the single round and the outgrown cells examined at later times for the frequency and type of septation. Under the conditions of the experiment, central division septation was blocked if HPUra (20 μm) was added before 70% (approximately) of the chromosome was replicated. Using higher concentrations of HPUra, 40 and 100 μm, it was shown that central division septation would occur at about its normal time if replication was blocked after this 70% stage but before termination. In these circumstances there was a distinct tendency for the DNA to remain close to the septum on both sides of it. The B. subtilis spore contains a single chromosome, which means that the central septum that forms in the absence of termination must pass through a partially completed chromosome. Electron microscopic evidence for such a situation has already been described (Van Iterson &; Aten, 1976). It is concluded that, at least under the restrictive conditions of the present experiments, termination of chromosome replication is not obligatory for the formation of the division septum with which it is normally coupled.     

An immobilized fork as a termination of replication intermediate in Bacillus subtilis

The structure of a DNA intermediate associated with termination of chromosome replication in Bacillus subtilis and derived from a unique BamHI 24.8 X 10(3) base-pair (bp) region of the chromosome has been investigated. The intermediate has properties expected for a forked structure. Gel electrophoresis followed by Southern transfer and hybridization to cloned DNA has shown it to comprise single strands of 15.4 X 10(3) bp and 24.8 X 10(3) bp, in approximately equimolar amounts. After purification away from the bulk of chromosomal DNA, electron microscopy of the intermediate established that 15% of the DNA was present as branched molecules and a significant proportion (11 of 31) of these contained two arms of matching length. The average dimensions (best estimates) of this unique class of Y-shaped molecule were 9.5(+/- 0.3) X 10(3), 15.1(+/- 0.4) X 10(3) and 24.6 24.6(+/- 0.6) X 10(3) bp for the stem, arms and end-to-end length, respectively. These values are consistent with the single strand composition of the intermediate as found. Furthermore, hybridization of the single strands to DNA from known locations within the BamHI 24.8 X 10(3) bp region has established the orientation of the forked intermediate relative to the genetic map. The intermediate presumably reflects the immobilization of the clockwise replication fork within the 24.8 X 10(3) bp region, at a location approximately 15.4 X 10(3) bp from the right end.     

Characterization of a combined DNA initiation and cell division mutant of Bacillus subtilis.

Summary The temperature-sensitive mutation in Bacillus subtilis 168-134ts, a conditional lethal DNA initiation mutant, was transferred to the minicell producing strain, CU 403 div IV-B1, to study he relationship of DNA synthesis to cell division. Markers in the combined mutant were verified by transduction. DNA replication kinetics, genome location by autoradiography, and clonal analysis of cell division patterns during spore outgrowths were investigated. Growth of the double mutant at the restrictive temperature results in an impressive reduction of the percentage cell length covered by DNA grain clusters (60.2% at 30° C compared to 8.6% after 2 h at 45° C). The probability of a minicell producing division in double mutant clones is essentially the same at 30° C and during the initial 2–3 h growth at 45° C at which time lysis begins. Residual division at 45° C is attributable to processes initiated at 30° C. The CU 403 div IV-B1, 134ts, double mutant divides about 25% as frequently relative to growth as do wild type CU 403 clones when incubated at permissive temperature. This is approximately 15% greater division suppression than previously found in the CU 403 div IV-B1 mutant strain, and is presumably due to interactions of the mutant gene products both of which affect DNA.     

Two-dimensional restriction analysis of the Bacillus subtilis genome: gene purification and ribosomal ribonucleic acid gene organization.

With two-dimensional restriction enzyme analysis we have been able to cleave the Bacillus subtilis genome and resolve the resulting deoxyribonucleic acid (DNA) segments into discrete bands on agarose gels. A general procedure for gene purification has been developed by coupling multidimensional restriction analysis with a biological assay for gene detection. The organization of ribosomal ribonucleic acid (rRNA) genes was studied by hybridizing 16S and 23S rRNA probes to the two-dimensional DNA banding patterns.     

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.     

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...