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.     

Membrane enrichment of genetic markers close to the origin and terminus during the deoxyribonucleic acid replication cycle in Bacillus subtilis.

A temperature-sensitive Bacillus subtilis initiation mutant was used to achieve one cycle of synchronized deoxyribonucleic acid (DNA) replication. Markers near the origin of replication and the terminus were assayed for association with the cell membrane at intervals during the DNA replication cycle. DNA near the origin and terminus was found to be enriched in the membrane fraction throughout the DNA replication cycle. The magnitude of membrane enrichment or origin and terminus markers varied coincidentally, possibly as a consequence of incubating the cells at 45 degrees C.     

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.     

Association of the replication terminus of the Bacillus subtilis chromosome to the cell membrane.

A chromosomal segment containing several genetic markers, from metB to thyA, near the replication terminus is associated with the membranous structure of Bacillus subtilis, but markers adjacent to this region, lys, ura, and metC, are not.     

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.     

DNA and protein sequence conservation at the replication terminus in Bacillus subtilis 168 and W23.

Cloned DNA from the replication terminus region of Bacillus subtilis 168 was used to identify and construct a restriction map of the homologous region in B. subtilis W23. With this information, DNA from the terminus region of W23 was cloned and the sequence was determined for a 1,499-base-pair segment spanning the expected terC site. The position of the site was then located more precisely. Use of the cloned DNA from strain W23 as a probe for digests of DNA from exponentially growing cells of the same strain established the presence of the slowly migrating replication termination intermediate (forked DNA). The orientation and dimensions of the forked molecule were consistent with arrest of the clockwise fork at the terC site in W23, as has been shown to occur in strain 168. Thus, despite significant differences between the two strains, the same termination mechanism appears to be used. The DNA sequences spanning the terC site in strains 168 and W23 showed a high level of homology (90.2%) close to the site but very little at a distance of approximately 250 base pairs from the site in one particular direction. The overall sequence comparison emphasised the importance of the open reading frame for a 122-amino-acid protein adjacent to terC. Although there were 22 base differences in the open reading frames between the strains, the amino acid sequence of the encoded protein was completely conserved. It is suggested that the amino acid sequence conservation reflects a role for the protein in the clockwise fork arrest mechanism as proposed earlier (M.T. Smith and R.G. Wake, J. Bacteriol. 170:4083-4090, 1988).     

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.     

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.     

Sequence limits of DNA strands in the arrest replication fork at the Bacillus subtilis chromosome terminus.

The DNA sequence limits of the leading and lagging strands in the arrested clockwise replication fork at the terminus of the Bacillus subtilis chromosome have been investigated. On the basis of hybridization to synthetic oligonucleotides corresponding to known positions in the terminus region sequence it has been shown that neither the leading nor lagging strands, as they approach terC, traverse the distal inverted repeat, IRI. But a small fraction of the leading strands pass through the proximal inverted repeat, IRII. This is consistent with IRI being the functional inverted repeat in arresting the clockwise fork. But most of the forks appear to stop at least 100 nucleotides short of IRI, and at various positions extending over a distance of at least 100 nucleotides.     

Nonrandom cosmid cloning and prophage SP beta homology near the replication terminus of the Bacillus subtilis chromosome.

From a library of Bacillus subtilis DNA cloned with the Escherichia coli cosmid vector pHC79, 85 recombinant cosmids containing DNA from near the replication terminus, terC, were identified. The DNA inserts of these cosmids were confined to three regions of a 350-kilobase segment of the chromosome extending from the left end of the SP beta prophage to approximately 75 kilobases on the right of terC. All B. subtilis genes known to reside in this segment, as well as the portion of the SP beta prophage that is expressed early in the lytic cycle of the phage, appeared to be absent from the library. A region of SP beta homology distinct from the prophage and just to the left of terC was identified.     

A replication terminus located at or near a replication checkpoint of Bacillus subtilis functions independently of stringent control

We have examined a replication terminus (psiL1) located on the left arm of the chromosome of Bacillus subtilis and within the yxcC gene and at or near the left replication checkpoint that is activated under stringent conditions. The psiL1 sequence appears to bind to two dimers of the replication terminator protein (RTP) rather weakly and seems to possess overlapping core and auxiliary sites that have some sequence similarities with normal Ter sites. Surprisingly, the asymmetrical, isolated psiL1 site arrested replication forks in vivo in both orientations and independent of stringent control. In vitro, the sequence arrested DnaB helicase in both orientations, albeit more weakly than the normal Ter1 terminus. The key points of mechanistic interest that emerge from the present work are: (i) strong binding of a Ter (psiL1) sequence to RTP did not appear to be essential for fork arrest and (ii) polarity of fork arrest could not be correlated in this case with just symmetrical protein-DNA interaction at the core and auxiliary sites of psiL1. On the basis of the result it would appear that the weak RTP-L1Ter interaction cannot by itself account for fork arrest, thus suggesting a role for DnaB-RTP interaction.     

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.     

Sequence analysis of replication origin of plasmid pLS11 of Bacillus subtilis IFO 3022.

The structure of a 1.6-kb SphI-HindIII DNA sequence necessary and sufficient for the replication of a 8.6-kb plasmid pLS11 of Bacillus subtilis IFO 3022, which is responsible for gamma-polyglutamate production, has been characterized by using a trimethoprim (Tmp)-resistance gene derived form B. subtilis TTK24 chromosomal DNA as a selective marker. The 1.6-kb DNA sequence contains a rep gene encoding the protein (333 amino acids) essential for initiation of replication and a possible origin of replication. The predicted REP protein of pLS11 has an overall homology with the REP proteins of pUH1 (74.8% identity), pBAA1 (92.8%), and pFTB14 (78.7%) in Bacillus spp., pLP1 (42.1%) and pLAB1000 (36.3%) in Lactobacillus spp., and pUB110 (35.3%) and pC194 (37.4%) in Staphylococcus aureus, but has not any similarity with the REP protein of the staphylococcal plasmid pT181.     

Functional analysis of the leading strand replication origin of plasmid pUB110 in Bacillus subtilis.

Supercoiled plasmid DNA is the substrate for initiation of pUB110 replication, and - by inference - for binding of its initiator protein (RepU) to the plasmid replication origin (oriU) in vivo. No hairpin structure is required for RepU-oriU recognition. RepH (the pC194 replication initiation protein) failed to initiate replication in trans at oriU. The nucleotides that determine the specificity of the replication initiation process are located within oriU but termination is unefficient. Therefore the segment that forms the full recognition signal for termination is probably located 3' of the oriU recognition sequence. Two overlapping domains, one for initiation and one required for termination, compose the leading strand replication origin of plasmid pUB110.     

Variations and coding features of the sequence spanning the replication terminus of Bacillus subtilis 168 and W23 chromosomes.

In a comparative study of the sequences of the 3-kb regions of DNA spanning the replication terminus, terC, of Bacillus subtilis strains 168 and W23, it was found that the latter contained an insertion of a large open reading frame (ORF405) whose translated protein product is a member of the cytochrome P-450 family. The insertion was about 34 nucleotides upstream from a putative promoter for the rtp gene. The sequenced regions contained a number of other ORFs. The translation product of one (ORF238) is a member of a previously identified oxidoreductase superfamily. The translation product of another (ORF257) is significantly similar to the proC product of Escherichia coli, but this ORF does not code for a functional proC product of B. subtilis.     

The Bacillus subtilis small cytoplasmic RNA gene and ''dnaX'' map near the chromosomal replication origin.

Summary TheBacillus subtilis small cytoplasmic RNA (scRNA) has an important, although not yet defined function in protein biosynthesis. Here we describe the mapping of the single copy scRNA gene and the flanking homolog todnaZX ofEscherichia coli, termed dnaX. The scRNA gene region of aB. subtilis wild-type strain was marked with acat gene and mapped by scoring chromosomal co-transformation rates of various mutant strains to chloramphenicol resistance and loss of the mutant phenotypes, respectively. This analysis, together with anEcoRI map comparison, places the scRNA gene anddnaX in the vicinity ofrecM near the replication origin region ofB. subtilis.     

Relocation of the replication terminus, terC, of Bacillus subtilis to a new chromosomal site

The terC-deletion strain of Bacillus subtilis 168, SU153 [Iismaa and Wake, J. Mol. Biol. 195 (1987) 299-310] was used for the re-insertion of a 1.75-kb segment of DNA containing terC at a site approx. 25 kb from its original position. The relocated terC in the new strain, SU160, was oriented normally with respect to the approaching clockwise replication fork, and was positioned such that this fork was the first to reach it. The relocated terC was effective in causing arrest of the clockwise fork, as evidenced by the appearance of a unique DNA species with a characteristic mobility in agarose gel electrophoresis and with a predicted single-strand composition. Thus, the previously cloned 1.75-kb terC-containing segment [Smith et al., Gene 38 (1985) 9-17] has not been altered with respect to TerC function and contains sufficient sequence for this function. The findings reported here provide the opportunity for establishing the minimal and essential sequence features of terC, and for examining its possible polarity of action in causing fork arrest.     

Coupling of asymmetric division to polar placement of replication origin regions in Bacillus subtilis

Entry into sporulation in Bacillus subtilis is characterized by the formation of a polar septum, which asymmetrically divides the developing cell into forespore (the smaller cell) and mother cell compartments, and by migration of replication origin regions to extreme opposite poles of the cell. Here we show that polar septation is closely correlated with movement of replication origins to the extreme poles of the cell. Replication origin regions were visualized by the use of a cassette of tandem copies of lacO that had been inserted in the chromosome near the origin of replication and decorated with green fluorescent protein-LacI. The results showed that extreme polar placement of replication origin regions is not under sporulation control and occurred in stationary phase under conditions under which entry into sporulation was prevented. On the other hand, the formation of a polar septum, which is under sporulation control, was almost invariably associated with the presence of a replication origin region in the forespore. Moreover, cells in which the polar placement of origin regions was perturbed by deletion of the gene (smc) for the structural maintenance of chromosomes (SMC) protein were impaired in polar division. A small proportion ( approximately 1%) of the mutant cells were able to undergo asymmetric division, but the forespore compartment of these exceptional cells was generally observed to contain a replication origin region. Immunofluorescence microscopy experiments indicated that the block in polar division caused by the absence of SMC occurred at or prior to the step of bipolar Z-ring formation by the cell division protein FtsZ. A model is discussed in which polar division is under the dual control of sporulation and an event associated with the placement of a replication origin at the cell pole.     

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.