R.G. Wake

Circularity of the Bacillus subtilis chromosome and further studies on its bidirectional replication

Abstract Replicating, circular chromosomes of Bacillus subtilis have been displayed by autoradiography of samples obtained from thymine-requiring spores germinating in the presence of [ 3 H]thymine and proceeding into the second round of replication. The observed chromosomes have the expected Cairns-type configuration, and the measured length of a completed (non-replicating) chromosome is 900 to 1100 μm. The extent to which bidirectional replication can proceed following initiation of a round has been investigated further. Thymine-requiring spores germinating in the presence of [ 3 H]thymidine were diluted into higher specific activity medium during the first round of replication and autoradiographs prepared from samples obtained shortly afterwards. From the pattern of labelling within individual loops that become visible, it is clear that replication can proceed in a bidirectional manner up to a stage that would account for replication of at least 50% of the whole chromosome. And, until this stage, the replication rates in both directions are approximately equal.

Identification of the replication terminator protein binding sites in the terminus region of theBacillus subtilis chromosome and stoichiometry of the binding

DNase I footprinting of the interaction between the replication terminator protein (RTP) of Bacillus subtilis and the inverted repeat region (IRR) at the chromosome terminus, to which it binds to block the clockwise replication fork, showed that two major regions of 41 base pairs (bp) were protected from cleavage. These regions corresponded approximately to the imperfect inverted repeats (IRI and IRII) identified previously. Band retardation analyses of the interaction between RTP and portions of the IRR established that each inverted repeat (IRI or IRII) contained two RTP binding sites. By sedimentation equilibrium in the ultracentrifuge, RTP was found to exist as a dimer of 29 kDa at neutral pH and concentrations above 0.2 g/l. Quantitative studies of the RTP-IRR interaction using [3H]RTP and [32P]IRR showed that the fully saturated complex contained eight RTP monomers per IRR. It is concluded that a dimer of RTP binds to each of the four sites in IRR. The apparent dissociation constant for the interaction was estimated (in the presence of 50% glycerol) to be 1.2 x 10(-11) M (dimer of RTP). Glycerol was found to have a marked effect on the affinity of RTP for the IRR and on the relative amounts of the interaction complexes formed; in the absence of glycerol the dissociation constant was approximately 50-fold higher and there was pronounced co-operative binding of RTP dimers to adjacent sites in each inverted repeat. Examination of the DNA sequence in IRI and IRII identified two 8 bp direct repeats in each. The regions protected from DNase I cleavage in each inverted repeat and the protection afforded by a core sequence spanning just one of the 8 bp direct repeats were consistent with each 8 bp repeat representing a recognition sequence for the RTP dimer. A model describing the binding of RTP to the IRR is presented.

Bidirectional chromosome replication in Bacillus subtilis

Abstract We have examined autoradiographically the pattern of DNA replication following the germination of Bacillus subtilis spores in [ 3 H]thymidine. Thymine-requiring spores were germinated in low specific activity medium and diluted into higher specific activity medium soon after the first round of replication was expected to start. After a further short period, replication was stopped and the chromosomal structures examined by autoradiography. From the pattern of labelling within individual replicating loops it is clear that the majority (≥75%) expand by growth at two positions that are opposite, i.e. expand bidirectionally. The loops continue to expand at approximately equal rates in both directions until at least 20% of the chromosome has been replicated. From a consideration of the other structural forms that become visible, it seems likely that most chromosomes replicate bidirectionally.

Structure of the RTP-DNA complex and the mechanism of polar replication fork arrest

The coordinated termination of DNA replication is an important step in the life cycle of bacteria with circular chromosomes, but has only been defined at a molecular level in two systems to date. Here we report the structure of an engineered replication terminator protein (RTP) of Bacillus subtilis in complex with a 21 base pair DNA by X-ray crystallography at 2.5 Å resolution. We also use NMR spectroscopic titration techniques. This work reveals a novel DNA interaction involving a dimeric winged helix domain protein that differs from predictions. While the two recognition helices of RTP are in close contact with the B-form DNA major grooves, the wings and N-termini of RTP do not form intimate contacts with the DNA. This structure provides insight into the molecular basis of polar replication fork arrest based on a model of cooperative binding and differential binding affinities of RTP to the two adjacent binding sites in the complete terminator.

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.

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

Abstract 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 μ m ) was added before 70% (approximately) of the chromosome was replicated. Using higher concentrations of HPUra, 40 and 100 μ m , 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.

The normal replication terminus of the Bacillus subtilis chromosome, terC, is dispensable for vegetative growth and sporulation☆

The Bacillus subtilis strains CU1693, CU1694 and CU1695 were shown by hybridization analysis to carry large deletions of the terminus region that originated within discrete fragments of the SP beta prophage genome. The absence of terC in CU1693 was demonstrated definitively by the identification of a novel junction fragment comprising SP beta DNA and DNA that lies on the other side of terC in the parent strain. This represented the deletion of approximately 230 kb of CU1693 DNA, with the removal of approximately 150 kb to the left of terC and approximately 80 kb to the right of terC. The lack of hybridization of CU1694 and CU1695 DNA to cloned DNA carrying the terC sequence and to cloned DNAs flanking terC suggested that terC is absent from the chromosome of each of these strains also, and that the deletions in CU1694 and CU1695 extend beyond the segment of the terminus region that has been mapped and cloned. The normal growth rate and morphology of CU1693, CU1694 and CU1695 relative to the parent strain when grown in complex medium indicated dispensability of terC for vegetative growth and division. B. subtilis SU153 was constructed using a specific deletion-insertion vector that was designed to effect the deletion of 11.2kb of DNA spanning terC, with the removal of approximately 9.7kb to the left of terC and approximately 1.kb to the right of terC. This manipulation did not introduce any readily detectable auxotrophic requirement. Physiological characterization of SU153 confirmed the dispensability of terC for vegetative growth and cell division, and also established the lack of requirement of terC for the specialized cell division that is associated with formation of the bacterial endospore.

Sequential replication of DNA in synchronously germinating Bacillus subtilis spores

Abstract Cairns (1963) has shown by autoradiography that when the E. coli chromosome, consisting of a single piece of DNA, replicates it does so by forming a “fork” which then moves along its length until the entire structure is doubled. Furthermore the results of Nagata (1963) and Yoshikawa and Sueoka (1963) show that in E. coli K12 (Hfr) and B. subtilis the starting point of replication must always be at the same position, with replication then proceeding in always the same direction along the length of the chromosome. This report describes the change in relative frequency of two unlinked genetic markers during one replication of the DNA in synchronously germinating B. subtilis spores. The results provide independent evidence for the general scheme suggested above. The synchrony in chromosome replication which accompanies emergence of the vegetative cell makes the germinating spore an attractive system for studying the events leading up to the initiation of DNA replication in bacteria.

Visualization of reinitiated chromosomes in Bacillus subtilis

Abstract Autoradiography has been used to display the replicated portion of both non-reinitiated and reinitiated chromosomes of Bacillus subtilis. The chromosomes had been partially replicated in the presence of [3H]thymine after an initial period of thymine starvation following germination of spores. In the case of non-reinitiated chromosomes, the replicated portion appears as a simple, closed loop, as expected for a Cairns-type replicating circle. The main type of reinitiated structure identified consists of a closed loop bifurcated in two regions into minor loops of approximately equal size. The pattern of linear-grain densities within the latter is consistent with their representing the material replicated as a result of reinitiation. The structures are considered to result from symmetrical reinitiations at chromosome origins. A scheme to account for the pathway of formation of the symmetrically reinitiated structures involves bidirectional replication, at least in the vicinity of the initiation site.

Completion of the replication and division cycle in temperature-sensitive DNA initiation mutants of Bacillus subtilis 168 at the non-permissive temperature

Abstract Spores of the temperature-sensitive DNA initiation mutants of Bacillus subtilis 168, TsB134 and dna -1(Ts), were allowed to germinate at 34 °C in the presence of [ 3 H]thymine until after the start of the first round of replication. The [ 3 H]-thymine was then replaced by non-radioactive thymine and the outgrowing spores transferred to a higher temperature (49 °C for TsB134, 45 °C for dna -1(Ts)) which had been shown to block completely the initiation of a second round of replication. Autoradiography of the colonies which developed under such conditions showed the majority to contain two grain clusters. In most cases the clusters were separated by a division septum. Thus, it appears that the temperature sensitive activity of the dna gene product in each case is not needed for either replication through the termination region of the chromosome or the ensuing segregation of the daughters. Further studies of the septation process showed that, when replication of the first round after germination was allowed to proceed to termination at the non-permissive temperature, a centrally located septum appeared readily in both mutants. On the other hand, at levels of thymine which prevented progress of the round to termination within the time of the experiment, central septation did not occur in colonies of the same length. Rather, asymmetrical septation occurred at a relatively low frequency. It appears that the formation of the central septum is coupled to termination and reflects normal division septation at the non-permissive temperature. It is concluded that in neither mutant does such septation require the action of the temperature-sensitive dna gene product at a late stage in the overall cycle.