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.     

Nucleotide sequence of the Bacillus subtilis phoR gene.

The nucleotide sequence of phoR, the positive and negative regulatory gene for alkaline phosphatase and phosphodiesterase formation in Bacillus subtilis, was determined. The sequence data predicted an open reading frame of 1,740 base pairs (579 amino acids) which overlaps the 5 base pairs of the preceding phoP coding sequence. The deduced amino acid sequence was significantly homologous with that of the Escherichia coli phoR gene product, which is the sensory element for the pho regulon.     

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.     

Replication origin region of Bacillus subtilis chromosome contains two rRNA operons.

The first replicating DNA fragment (BamHI-7) of the Bacillus subtilis chromosome contains two promoters for a rRNA operon. A map of restriction enzyme cleavage sites of the region of replication origin suggests the presence of a second rRNA operon in this region. Hybridization of rRNA genes (rDNA) with DNA fragments derived from the origin region by treatment with various enzymes clearly revealed two rRNA operons in this region, one at the B7-B3 junction and the other at the B5-B6 junction. The restriction enzyme cleavage sites surrounding the rRNA operons show that the operon at the B5-B6 junction corresponds to the rrnA operon. A novel operon at the B7-B3 junction was termed rrnO. Transformation by density-labeled fragments of the origin region showed that the first replicating marker, guaA, is located in the B3 fragment. From these results, a map was constructed for the first time to correlate the genetic markers with the physical structure of the replication origin region of the B. subtilis chromosome. The role of the rrnO operon in regulating the initiation of chromosomal replication is discussed, based on the fact that the promoter of the rrnO operon suppresses the replication of the plasmid carrying the promoter.     

Impediment to replication fork movement in the terminus region of the Bacillus subtilis chromosome

The terminus regions of the chromosomes of three strains of Bacillus subtilis 168 were radioactively labelled by supplying [3H]thymine towards the end of a round of replication. These strains lacked or contained the prophage SP beta c2. Following restriction endonuclease digestion of the purified DNA and fluorography, an SP beta c2-related perturbation of the terminus-labelling profile was observed, which was completely consistent with the previously suggested existence of an impediment to replication fork movement (terC) within a BamHI 24.8 X 10(3) base fragment (Weiss & Wake, 1983). The present data suggest that terC is located within the 11.4 X 10(3) base BamHI + SalI double-digest portion of this BamHI fragment.     

Nucleotide sequence of threonine tRNA from Bacillus subtilis.

A threonine tRNA was purified from Bacillus subtilis W168 by a combined use of column chromatographic systems. The nucleotide sequence was determined to be pG-C-C-G-G-U-G-U-A-G-C-U-C-A-A-U-D-G-G-D(U)-A-G-A-G-C-A-A-C-U-G-A-C-U-mo5U-G-U-t6A-A-psi-C-A-G-U-A-G-m7G-U-U-G-G-G-G-G-T-psi-C-A-A-G-U-C-C-U-C-U-U-G-C-C-G-G-C-A-C-C-AOH, where about 40 % of D20 remained unmodified as U20. It consists of 76 nucleotides including a new minor nucleoside, 5-methoxyuridine (mo5U), which occupies the wobble position of anticodon.     

Nucleotide sequence of the Bacillus subtilis tryptophan operon

In Bacillus subtilis, tryptophan biosynthesis is one of the most thoroughly characterized biosynthetic pathways. Recombinant DNA methodology has permitted a rapid characterization of the tryptophan (trp) gene cluster at the molecular level. In this report the nucleotide sequence of the six structural genes together with the intercistronic regions and flanking regulatory regions are presented.     

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome

Summary A BamHI restriction endonuclease fragment, B7, which is replicated first among all other fragments derived from the Bacillus subtilis chromosome, was cloned in Escherichia coli using as vector a hybrid plasmid pMS102 that can replicate both in E. coli and B. subtilis. Digestion of pMS102 with BamHI produced two fragments and the smaller one was replaced by the B7 fragment.The cloned plasmid pMS102-B7 exhibited some peculiar properties that were not observed with plasmids containing other fragments from the B. subtilis chromosome. (1) E. coli cells harboring this plasmid stuck to each other and to glass. This property was more apparent when cells were grown in poor media. (2) E. coli cells tended to lose the plasmid spontaneously when they were grown without the selective pressure favorable to the plasmid. (3) The frequency of transformation of B. subtilis by pMS102-B7 was less than 1/1,000 of that by the vector plasmid pMS102. The number of copies of pMS102-B7 present in the transformants was also markedly reduced, although the pUB110 origin of replication on the vector was intact and should be functional in B. subtilis. This inhibitory effect of the B7 fragment on plasmid replication was confirmed more directly by developing a semi in vitro replication system using protoplasts.Both in E. coli and B. subtilis the B7 fragment affected replication of its own molecule but not that of the coexisting plasmid with an identical replication system. The implication of the function of the B7 fragment in the initiation of the B. subtilis chromosome will be discussed.     

Nucleotide sequence of a lysine tRNA from Bacillus subtilis.

A lysine tRNA (tRNA1Lys) was purified from Bacillus subtilis W168 by a consecutive use of several column chromatographic systems. The nucleotide sequence was determined to be pG-A-G-C-C-A-U-U-A-G-C-U-C-A-G-U-D-G-G-D-A-G-A-G-C-A-U-C-U-G-A-C-U-U(U*)-U-U-K-A-psi-C-A-G-A-G-G-m7G(G)-U-C-G-A-A-G-G-T-psi-C-G-A-G-U-C-C-U-U-C-A-U-G-G-C-U-C-A-C-C-AOH, where K and U* are unidentified nucleosides. The nucleosides of U34 and m7G46 were partially substituted with U* and G, respectively. The binding ability of lysyl-tRNA1Lys to Escherichia coli ribosomes was stimulated with ApApA as well as ApApG.