Cloning and localization of the Bacillus subtilis chromosome replication terminus, terC

A 10.9-kb segment of the Bacillus subtilis 168 chromosome has been cloned in an Escherichia coli plasmid and shown to contain terC (the replication terminus of the chromosome). The terC-containing portion of this plasmid has been subcloned within each of two overlapping fragments of DNA, 1.75 and 1.95 kb, again in E. coli plasmids. These have afforded a more precise definition of the location of terC in the B. subtilis chromosome and provided material for a detailed analysis of the structure and functioning of this site.     

Specific labeling of the Bacillus subtilis chromosome terminus

The deoxyribonucleic acid labeled by a procedure described previously for labeling the chromosomal terminus of B. subtilis 168 was substantially enriched for sequences homologous to bacteriophages SP beta and phi 3T, which integrate in the terminal region.     

Replication terminus of the Bacillus subtilis chromosome.

Bidirectional replication of the Bacillus subtilis chromosome terminates at a point on the circular chromosome which is symmetrically opposite to the replication origin. Since replication rates are similar in both "halves" of the chromosome, termination presumably occurs at the meeting point of the two replication forks. To investigate whether the DNA sequence of this region of the chromosome contributes to the termination event, we have determined the latest replicating region of a chromosome in which this DNA sequence is no longer symmetrically opposite to the origin. The merodiploid strain GSY1127 has a very large nontandem duplication (approximately 25% of the total chromosome length) in the left-hand half of the chromosome, so that size and symmetry of this chromosome are grossly different from those of normal strains. We have examined the replication order of genetic markers in this strain by measuring subtilis terminal marker for replication remains a terminal marker in the merodiploid, i.e., replicates later than a marker situated symmetrically opposite to the replication origin. These results were supported by replication orders determined by pulse-density transfer experiments during synchronous replication. The data obtained indicate that there is a preferred site for the termination of replication in the B. subtilis chromosome.     

Highly specific labeling of the Bacillus subtilis chromosome terminus

By making use of the sporulation process, the terminus region of the Bacillus subtilis chromosome has been labeled with [3H]thymine in a highly specific manner. The result achieved supports the view that B. subtilis spores contain only completed chromosomes.     

Cellular location of origin and terminus of replication in Bacillus subtilis.

The origin of replication of Bacillus subtilis 168 trp thy dna-1 (temperature-sensitive initiation mutant) was labeled with [3H]thymidine. Analysis of labeled cells by autoradiography revealed that most of the radioactivity was associated with cell pole areas. To label the terminus, cells that had initiated were treated with chloramphenicol to inhibit cell growth and division but to allow continued DNA synthesis. These cells were then labeled with [3H]thymidine at a time when chromosome replication was nearly complete. The distribution of radioactivity was similar to that observed in origin-labeled cells. In contrast, exponentially growing cells that were labeled for a brief time at the permissive temperature showed a random distribution of radioactivity. These data indicate that the origin and terminus of replication are located at cell poles.     

Structural and functional properties of a Bacillus subtilis temperature-sensitive sigma(A) factor

Bacillus subtilis DB1005 is a temperature-sensitive (Ts) sigA mutant containing double-amino-acid substitutions (I198A and I202A) on the hydrophobic face of the promoter -10 binding helix of sigma(A) factor. We have analyzed the structural and functional properties of this mutant sigma(A) factor both in vivo and in vitro. Our data revealed that the Ts sigma(A) factor possessed predominantly a multimeric structure which was prone to aggregation at restrictive temperature. The extensive aggregation of the Ts sigma(A) resulted in a very low core-binding activity of the Ts sigma(A) factor and a markedly reduced sigma(A)-RNA polymerase activity in B. subtilis DB1005, suggesting that extensive aggregation of the Ts sigma(A) is the main trigger for the temperature sensitivity of B. subtilis DB1005. Partial proteolysis, tryptophan fluorescence and 1-anilinonaphthalene-8-sulfonate-binding analyses revealed that the hydrophobic face of the promoter -10 binding helix and also the hydrophobic core region of the Ts sigma(A) factor were readily exposed on the protein surface. This hydrophobic exposure provides an important cue for mutual interaction between molecules of the Ts sigma(A) and allows the formation of multimeric Ts sigma(A). Our results also indicate that Ile-198 and Ile-202 on the hydrophobic face of the promoter -10 binding helix are essential to ensure the correct folding and stabilization of the functional structure of sigma(A) factor.     

Differential and dynamic localization of topoisomerases in Bacillus subtilis

Visualization of topoisomerases in live Bacillus subtilis cells showed that Topo I, Topo IV, and DNA gyrase differentially localize on the nucleoids but are absent at cytosolic spaces surrounding the nucleoids, suggesting that these topoisomerases interact with many regions of the chromosome. While both subunits of Topo IV were uniformly distributed throughout the nucleoids, Topo I and gyrase formed discrete accumulations, or foci, on the nucleoids in a large fraction of the cells, which showed highly dynamic movements. Three-dimensional time lapse microscopy showed that gyrase foci accumulate and dissipate within a 1-min time scale, revealing dynamic assembly and disassembly of subcellular topoisomerase centers. Gyrase centers frequently colocalized with the central DNA replication machinery, suggesting a major role for gyrase at the replication fork, while Topo I foci were frequently close to or colocalized with the structural maintenance of chromosomes (SMC) chromosome segregation complex. The findings suggest that different areas of supercoiling exist on the B. subtilis nucleoids, which are highly dynamic, with a high degree of positive supercoiling attracting gyrase to the replication machinery and areas of negative supercoiling at the bipolar SMC condensation centers recruiting Topo I.     

Attachment of the chromosomal terminus of Bacillus subtilis to a fast-sedimenting particle

After gently lysed protoplasts of exponential phase cells of Bacillus subtilis were treated with restriction endonuclease BamHI, 99% of the DNA did not sediment with the plasma membrane. This DNA was fractionated on sucrose gradients into (i) a fast-sedimenting fraction highly enriched for genes from the origin and terminus (purA and ilvA), (ii) a 50 to 100S component also enriched for purA and ilvA, and (iii) the bulk of the DNA. The fast-sedimenting fraction was dissociated by Sarkosyl; this fraction contained a substantial amount of protein and is probably a membrane subparticle. The S value of the 50 to 100S component was not greatly affected by Sarkosyl treatment, but these particles were unable to penetrate an agarose gel during electrophoresis and were retained by nitrocellulose filters. The terminus DNA in the fast-sedimenting fraction and the 50 to 100S component contained a large restriction fragment (1.5 x 10(7) to 2.0 x 10(7) daltons) encoding ilvA, thyB, and ilvD. The bulk of the SP beta prophage and metB, which lie to the right and left, respectively, of the ilvA-ilvD cluster, were not part of the complex. citK, which lies to the right of SP beta, appeared to be present in the fast-sedimenting complexes. The neighboring genes kauA and gltA were not part of the fast-sedimenting complexes. The presence of terminus DNA in the fast-sedimenting components was also demonstrated by a radiochemical method.     

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.     

Purification and properties of mannanase from Bacillus subtilis

Extracellular mannanase from Bacillus subtilis NM-39, an isolate from Philippine soil, was purified about 240-fold with a yield of 7.3% by ammonium sulphate fractionation, DEAE-Toyopearl chromatography and Sephacryl S-200 gel filtration. Its M _r was 38 kDa and it had a pI of 4.8 and optimum activity at pH 5.0 and 55°C. It was stable at pH 4 to 9 and below 55°C. The amino acid composition of the enzyme was in the order Gly>Glx>Ser and Asx>Ala.N.S. Mendoza and L.M. Joson are with Industrial Technology Development Institute, Department of Science and Technology, Manila, Philippines. M. Arai and T. Kawaguchi are with Department of Agricultural Chemistry, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka 593, Japan; T. Yoshida is with Faculty of Engineering, Osaka University, Suita, Osaka 565, Japan.     

Correlation Between Pigment Production and Amino Acid Requirements in Bacillus subtilis

Several Bacillus subtilis W-23 auxotrophs were unable to produce wild-type pigment normally on minimal agars supplemented sufficiently for growth. This offers a reliable means for scoring genotypes.     

n-Terminal Amino Acid Sequences of Neutral Proteases from Bacillus amyloliquefaciens and Bacillus subtilis: Identification of a Neutral Protease Gene Cloned in Bacillus subtilis

Bacillus subtilis 1A20 transformed with a hybrid plasmid, pNP150, to which a DNA fragment from Bacillus amyloliquefaciens F was attached, produced a large amount of a neutral protease. To identify the origin of the gene specifying this neutral protease, neutral proteases from B. amyloliquefaciens F, B. subtilis NP58 (a derivative of Marburg 6160), and B. subtilis 1A20 transformed with pNP150 were purified. We investigated their immunological properties and primary structures.

The proteases from these two species were indistinguishable by chromatography, but they were distinguishable from each other by SDS-polyacrylamide gel electrophoresis and double immunodiffusion. Amino acid sequencing of these two proteases by Edman degradation showed that there were four substitutions in the 20-residue amino acid sequence from the N-termini.

Neutral protease from the transformant had the same immunological characteristics and N-terminal amino acid sequence as that from B. amyloliquefaciens. These results meant that the gene in question was derived from a gene specifying the neutral protease in this bacterium.     

Bacillus subtilis DEAD protein YdbR possesses ATPase, RNA binding, and RNA unwinding activities

The product of an open reading frame (ORF) (called YdbR) identified while analyzing the Bacillus subtilis genome has been classified as an Asp-Glu-Ala-Asp (DEAD) protein, but the biological function and enzymology of YdbR have not been characterized in detail. Here we show that recombinant YdbR-His(6) purified from Escherichia coli is an ATP-independent RNA binding protein. It also possesses RNA-dependent ATPase activity stimulated not only by total RNA from B. subtilis but also by an RNA that is irrelevant to that of B. subtilis. Functional analysis indicated that the growth rate of a DeltaydbR mutant strain of B. subtilis was reduced as compared with that of the wild type not only at 37 degrees C, but more severely at 22 degrees C.     

Restriction map of DNA spanning the replication terminus of the Bacillus subtilis chromosome

The Bacillus subtilis 168 dna-1 chromosome was labelled during sporulation with [3H]thymine for five minutes immediately before termination of replication. The isolated radioactive DNA was cleaved with BamHI (or SalI) and the resulting restriction fragments separated by agarose gel electrophoresis. The individual fragments, fractionated into a series of slices cut from the gel, were then cleaved with SalI (or BamHI) and the double-digest fragments identified by electrophoresis and fluorography. All major fragments and most minor ones present in a whole double-digest were assigned to BamHI and SalI parents. Such information enabled the construction of an unambiguous restriction map of 150 X 10(3) bases of the approximately 250 X 10(3) bases of DNA labelled in the five minutes. In conjunction with published data on the order of replication of restriction fragments as termination is approached, it was clear that most (105 X 10(3) bases) of the mapped DNA was replicated by a major fork moving in one direction towards a BamHI 24.8 X 10(3) base fragment. The 45 X 10(3) bases extending to the other side of this region were labelled only slightly, and presumably was replicated by a fork that approached the other in an opposite direction until its progress was blocked or severely impeded within this region at a site, referred to as terC, sometime (less than 5 min) earlier. The regions of the map replicated in the final 2.5 and 1.0 minute by the major fork were also identified.