Subcellular positioning of the origin region of the Bacillus subtilis chromosome is independent of sequences within oriC, the site of replication initiation, and the replication initiator DnaA

Regions of bacterial chromosomes occupy characteristic locations within the cell. In Bacillus subtilis, the origin of replication, oriC, is located at 0 degrees /360 degrees on the circular chromosome. After duplication, sister 0 degrees regions rapidly move to and then reside near the cell quarters. It has been hypothesized that origin function or oriC sequences contribute to positioning and movement of the 0 degrees region. We found that the position of a given chromosomal region does not depend on initiation of replication from the 0 degrees region. In an oriC mutant strain that replicates from a heterologous origin (oriN) at 257 degrees , the position of both the 0 degrees and 257 degrees regions was similar to that in wild-type cells. Thus, positioning of chromosomal regions appears to be independent of which region is replicated first. Furthermore, we found that neither oriC sequences nor the replication initiator DnaA is required or sufficient for positioning a region near the cell quarters. A sequence within oriC previously proposed to play a critical role in chromosome positioning and partitioning was found to make little, if any, contribution. We propose that uncharacterized sites outside of oriC are involved in moving and/or maintaining the 0 degrees region near the cell quarters.     

A large dispersed chromosomal region required for chromosome segregation in sporulating cells of Bacillus subtilis

The cis-acting sequences required for chromosome segregation are poorly understood in most organisms, including bacteria. Sporulating cells of Bacillus subtilis undergo an unusual asymmetric cell division during which the origin of DNA replication (oriC) region of the chromosome migrates to an extreme polar position. We have now characterized the sequences required for this migration. We show that the previously characterized soj-spo0J chromosome segregation system is not essential for chromosome movement to the cell pole, so this must be driven by an additional segregation mechanism. Observations on a large set of precisely engineered chromosomal inversions and translocations have identified a polar localization region (PLR), which lies approximately 150-300 kbp to the left of oriC. Surprisingly, oriC itself has no involvement in this chromosome segregation system. Dissection of the PLR showed that it has internal functional redundancy, reminiscent of the large diffuse centromeres of most eukaryotic cells.     

Suppression of initiation defects of chromosome replication in Bacillus subtilis dnaA and oriC-deleted mutants by integration of a plasmid replicon into the chromosomes.

We constructed Bacillus subtilis strains in which chromosome replication initiates from the minimal replicon of a plasmid isolated from Bacillus natto, independently of oriC. Integration of the replicon in either orientation at the proA locus (115 degrees on the genetic map) suppressed the temperature-sensitive phenotype caused by a mutation in dnaA, a gene required for initiation of replication from oriC. In addition, in a strain with the plasmid replicon integrated into the chromosome, we were able to delete sequences required for oriC function. These strains were viable but had a slower growth rate than the oriC+ strains. Marker frequency analysis revealed that both pyrD and metD, genes close to proA, showed the highest values among the markers (genes) measured, and those of other markers decreased symmetrically with distance from the site of the integration (proA). These results indicated that the integrated plasmid replicon operated as a new and sole origin of chromosome replication in these strains and that the mode of replication was bidirectional. Interestingly, these mutants produced anucleate cells at a high frequency (about 40% in exponential culture), and the distribution of chromosomes in the cells was irregular. A change in the site and mechanism (from oriC to a plasmid system) of initiation appears to have resulted in a drastic alteration in coordination between chromosome replication and chromosome partition or cell division.     

Cloning of an autonomously replicating sequence (ars) from the Bacillus subtilis chromosome

Cloning of an autonomously replicating sequence (ars) from the origin region of Bacillus subtilis was previously unsuccessful because of the strong incompatibility exerted by sequences located within the oriC region. Using an ars searching vector which would be selective for drug resistance even at one copy per cell, and by cloning large fragments covering as much as possible of the oriC region, we have succeeded in isolating ars fragments from the origin region of the chromosome. The minimum essential fragment contains two DnaA-box regions (non-translatable regions containing multiple repeats of DnaA-box) separated by the dnaA gene. Neither one of the DnaA-box regions by itself showed ars activity. When constructed as oriC plasmids, the dnaA coding region could be removed without affecting ars activity. The minimum distance between the two DnaA-box regions obtained so far is 274 bp. The copy number of the oriC plasmid is estimated as one per replicating chromosome. These plasmids are unstable and tend to be lost or integrated into chromosome.     

MukB colocalizes with the oriC region and is required for organization of the two Escherichia coli chromosome arms into separate cell halves

The circular Escherichia coli chromosome is organized by bidirectional replication into two equal left and right arms (replichores). Each arm occupies a separate cell half, with the origin of replication (oriC) at mid-cell. E. coli MukBEF belongs to the ubiquitous family of SMC protein complexes that play key roles in chromosome organization and processing. In mukBEF mutants, viability is restricted to low temperature with production of anucleate cells, reflecting chromosome segregation defects. We show that in mukB mutant cells, the two chromosome arms do not separate into distinct cell halves, but extend from pole to pole with the oriC region located at the old pole. Mutations in topA, encoding topoisomerase I, do not suppress the aberrant positioning of chromosomal loci in mukB cells, despite suppressing the temperature-sensitivity and production of anucleate cells. Furthermore, we show that MukB and the oriC region generally colocalize throughout the cell cycle, even when oriC localization is aberrant. We propose that MukBEF initiates the normal bidirectional organization of the chromosome from the oriC region.     

Polar localization of the Escherichia coli oriC region is independent of the site of replication initiation

The location of the origin-linked region of the Escherichia coli chromosome was analysed in strains lacking the core origin locus, oriC. In these strains, which initiate replication from F factors integrated at different locations around the chromosome, origin-linked DNA remains localized near the cell poles, as in wild-type cells. In contrast, minichromosomes containing 7 kb of chromosomal DNA including oriC are generally excluded from the ends of the cell. Thus, we propose that positioning of the wild-type origins at the poles is not a function of their order of replication but a sequence-specific phenomenon. It is proposed that there are centromere-like sequences, bordering the wild-type origin of replication, which are used by host mechanisms to direct the proper placement of the origin region of the chromosome. This function, combined with other host processes, may assure efficient segregation of the E. coli chromosome.     

Subcellular localization of Dna-initiation proteins of Bacillus subtilis: evidence that chromosome replication begins at either edge of the nucleoids

We examined the intracellular distribution of Bacillus subtilis Dna-initiation proteins by immunofluorescence microscopy to visualize the initiation complex of replication in vivo. DnaA was distributed throughout the cytoplasm, but both DnaB and DnaI were always detected as foci during the cell-division cycle. Interaction of DnaI with the DnaC helicase by the yeast two-hybrid assay suggests that DnaI acts as a helicase loader. The number of DnaB and DnaI foci within the cell exceeded that of oriC. Although the foci were not always co-localized with oriC, they seemed to be localized near the outer or inner edges of the nucleoids at initiation of replication. When the replication cycle was synchronized in cells using a temperature-sensitive dnaA mutant, duplication of the oriC region was observed predominantly near an edge of the nucleoid. Before initiation occurred, each one of the DnaB and DnaI foci was frequently observed near there. Furthermore, DnaX-GFP (DnaX is a component of DNA polymerase III) foci were detected near either of the edges of the nucleoids at the onset of replication. These results suggest that the replisome is recruited into oriC near either edge of the nucleoids to initiate chromosome replication in B. subtilis.     

Structure and function of the region of the replication origin of the Bacillus subtilis chromosome. III. Nucleotide sequence of some 10,000 base pairs in the origin region.

Approximately 10,000 nucleotides were sequenced in the oriC region of the Bacillus subtilis chromosome. The first replicating DNA strands are hybridized with a SalI-EcoRI fragment (nucleotide #1206-2954) in one direction (left to right) and an EcoRI-PstI fragment (#2949-4233) in the other. Seven open reading frames (ORF) accompanied with Shine-Dalgarno (SD) sequences were identified. ORF638 and ORF821 were identified as gyrB and gyrA genes respectively based on genetic evidences and amino acid sequence data. Comparison of amino acid sequences revealed that ORF44, ORF446, ORF378 and ORF323 are homologous with rpmH, dnaA, dnaN and recF of Escherichia coli, respectively. Thus, the organization of the ORFs from ORF44 to ORF638 resembles the organization of genes in the rpmH-gyrB region of the E. coli chromosome. Two non-coding regions characteristic for oriC signals were found near the site of initiation of the first replicating DNA. They are composed of repeating sequences whose consensus sequence TTAT(C/A)CACA is identical to that of 4 repeating sequences in the oriC of E. coli.     

Characterization of the oriC region of Mycobacterium smegmatis.

A 3.5-kb DNA fragment containing the dnaA region of Mycobacterium smegmatis has been hypothesized to be the chromosomal origin of replication or oriC (M. Rajagopalan et al., J. Bacteriol. 177:6527-6535, 1995). This region included the rpmH gene, the dnaA gene, and a major portion of the dnaN gene as well as the rpmH-dnaA and dnaA-dnaN intergenic regions. Deletion analyses of this region revealed that a 531-bp DNA fragment from the dnaA-dnaN intergenic region was sufficient to exhibit oriC activity, while a 495-bp fragment from the same region failed to exhibit oriC activity. The oriC activities of plasmids containing the 531-bp sequence was less than the activities of those containing the entire dnaA region, suggesting that the regions flanking the 531-bp sequence stimulated oriC activity. The 531-bp region contained several putative nine-nucleotide DnaA-protein recognition sequences [TT(G/C)TCCACA] and a single 11-nucleotide AT-rich cluster. Replacement of adenine with guanine at position 9 in five of the putative DnaA boxes decreased oriC activity. Mutations at other positions in two of the DnaA boxes also decreased oriC activity. Deletion of the 11-nucleotide AT-rich cluster completely abolished oriC activity. These data indicate that the designated DnaA boxes and the AT-rich cluster of the M. smegmatis dnaA-dnaN intergenic region are essential for oriC activity. We suggest that M. smegmatis oriC replication could involve interactions of the DnaA protein with the putative DnaA boxes as well as with the AT-rich cluster.     

A fixed distance for separation of newly replicated copies of oriC in Bacillus subtilis: implications for co-ordination of chromosome segregation and cell division

The Spo0J protein of Bacillus subtilis is required for normal chromosome segregation and forms discrete subcellular assemblies closely associated with the oriC region of the chromosome. Here we show that duplication of Spo0J foci occurs early in the DNA replication cycle and that this requires the initiation of DNA replication at oriC but not elongation beyond the nearby STer sites. Soon after duplication, sister oriC /Spo0J foci move rapidly apart to achieve a fixed separation of about 0.7 μm, reminiscent of the segregation of eukaryotic chromosomes on the mitotic spindle. The magnitude of the fixed separation distance may explain how chromosome segregation is kept in close register with cell growth and the initiation mass for DNA replication. It could also explain how segregation can proceed accurately in the absence of cell division. The kinetics of focal separation suggest that one role of Spo0J protein may be to facilitate formation of separate sister oriC complexes that can be segregated.     

Cloning and characterization of the chromosomal replication origin region of Amycolatopsis mediterranei U32

The chromosomal replication origins (oriC) of gram positive, acid-fast actinomycetes have been investigated in streptomycetes and mycobacteria. A 1339 bp DNA fragment of the putative oriC region from the rifamycin SV producer Amycolatopsis mediterranei U32 was cloned by PCR amplification employing primers designed based on the conserved flanking genes of dnaA and dnaN. The 884 bp sequence of the intergenic region between dnaA and dnaN genes consists of 19 DnaA-boxes and two 13-mer AT-rich sequences, which is similar to the oriC structure of Streptomyces lividans. A mini-chromosome constructed by cloning the putative U32 oriC DNA fragment into an Escherichia coli plasmid was able to replicate autonomously, but was unstable, in A. mediterranei U32 with an estimated copy number of two per cell. Although efficient replication of the mini-chromosome in U32 requires the complete set of DnaA-boxes and AT-rich regions, only one of the AT-rich sequences together with part of the DnaA-boxes is sufficient, suggesting the presence of combinatorial alternatives for a functional oriC region of A. mediterranei U32. Phylogenetic analysis based on definite oriC sequences among eubacteria reflects well the relationship between these species.     

Conversion to bidirectional replication after unidirectional initiation from R1 plasmid origin integrated at oriC in Escherichia coli

The cell division phenotypes of Escherichia coli with its chromosome replication driven by oriR (from plasmid R1) were examined by fluorescence microscopy and flow cytometry. Chromosome replication patterns in these strains were followed by marker frequency analyses. In one of the strains, the unidirectional oriR was integrated so that the replication fork moved clockwise from the oriC region, and bacterial growth and division were similar to those of the wild-type parent. The bacteria were able to convert the unidirectional initiation from oriR into bidirectional replication. The site for conversion of uni- to bidirectional replication seemed to be localized and could be mapped genetically within 6 min to the immediate right of the minimal oriC . Replication starting in the counterclockwise direction from the R1 replicon integrated at the same site in the opposite orientation could not be described as either bi- or unidirectional, as no single predominant origin could be discerned from the more or less flat marker frequency pattern. These strains also showed extensive filamentation, irregular nucleoid distribution and the presence of anucleate cells, indicative of segregation and division defects. Comparison among intR1 derivatives differing in the position of the integrated oriR relative to the chromosome origin suggested that the oriC sequence itself was dispensable for the conversion to bidirectionality. However, passage of the replication fork over the 6 min region to the right of oriC seemed important for the bidirectional replication pattern and normal cell division phenotype.     

Genetic mapping of the chromosomal replication origin of Salmonella typhimurium.

Two hundred strains of Escherichia coli harboring Filv+ plasmids which carry a segment of the Salmonella typhimurium chromosome were isolated independently. Among them, two strains were found to harbor F' plasmids that are able to replicate in Hfr cells of E. coli; i.e., they carry a site designated poh (permissive on Hfr) of the S. typhimurium chromosome. The poh site is presumably identical with the replication origin (oriC) of the bacterial chromosome. These two plasmids carry the dnaA-uncA-rbs-ilv-cya-metE region of the chromosome of S. typhimurium. Other F' plasmids which only carried the ilv-cya-metE region were unable to be maintained in Hfr cells. The poh site (= oriC) of S. typhimurium thus is located in the uhp-ilv region of the chromosome. The two plasmids carrying the poh site of S. typhimurium can suppress the temperature-sensitive character of an E. coli mutant that carries the temperature-sensitive dnaA46 allele, when the plasmids exist in the mutant cells. This suggests that the dnaA chromosome in place of the dnaA gene product of E. coli itself. The ability of the plasmids carrying the poh site of S. typhimurium to replicate in Hfr cells of E. coli suggests that the replication system of E. coli can recognize the Salmonella replication origin.     

The bacterial chromosome segregation protein Spo0J spreads along DNA from parS nucleation sites

Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.     

In vitro assembly of a prepriming complex at the origin of the Escherichia coli chromosome

During initiation of DNA replication of plasmids containing the origin of the Escherichia coli chromosome (oriC), the proteins dnaA, dnaB, and dnaC interact and assemble a complex at oriC. The complex is larger and more asymmetric than that formed by dnaA protein and embraces an extra 50 base pairs at the left side of the minimal oriC sequence. Both dnaA and dnaB proteins have been identified in the complex by electron microscopy and antibody binding; dnaC protein was not detected. HU protein, which stimulates the activity of the initiation reaction, was often present. Entry of dnaB protein required dnaA and dnaC proteins and a supercoiled template. Thus, a complex structure, involving multiple proteins and a large region of DNA, must be formed at the origin to prepare the template for priming and replication.     

The origin of replication, oriC, and the dnaA protein are dispensable in stable DNA replication (sdrA) mutants of Escherichia coli K-12

The sdrA224 mutants of Escherichia coli K-12, capable of continued DNA replication in the absence of protein synthesis (stable DNA replication), tolerate inactivation of the dnaA gene by insertion of transposon Tn10. Furthermore, oriC, the origin of E. coli chromosome replication, can be deleted from the chromosome of sdrA mutants without loss of viability. The results suggest the presence of a second, normally repressed, initiation system for chromosome replication alternative to the 'normal' dnaA+ oriC+-dependent initiation mechanism.     

Chromosome Partitioning in Escherichia coli in the Absence of Dam-Directed Methylation

Escherichia coli dam mutants, lacking the GATC DNA methylase, do not produce anucleate cells at high frequencies, suggesting that hemimethylation of the chromosome origin of replication, oriC, is not essential for correct chromosome partitioning.     

Evidence for the involvement of the 16kD gene promoter in initiation of chromosomal replication of Escherichia coli strains carrying a B/r-derived replication origin.

Initiation of chromosomal DNA replication of several Escherichia coli dnaA (Ts) strains is diminished in cell harbouring pBR322 hybrid plasmids carrying both oriC and the adjacent 16kD gene promoter of E. coli K12. This perturbance, resulting in very slow growth, is caused both by the dnaA allele and the E. coli B/r-derived region of the replication origin of these strains. Cloning and DNA sequence analysis of the E. coli B/r replication origin revealed several base differences as compared to the E. coli K12 sequence. The replication origin of temperature sensitive fast growing mutants, originating from a homologous exchange between chromosomal and plasmid DNA sequences were also cloned. Sequence data showed that a single base change within the promoter of the 16kD gene of these dnaA (Ts) strains is able to suppress the inhibition of chromosomal DNA replication by the mentioned pBR322 hybrid plasmids. Our results strongly indicate a role of the 16kD gene promoter in control of initiation of chromosomal DNA replication.     

The replicated ftsQAZ and minB chromosomal regions of Escherichia coli segregate on average in line with nucleoid movement

The average cellular positions of the ftsQAZ region (2 min) and the minB region (26.5 min) during the cell cycle was determined by fluorescent in situ hybridization using the position of oriC as a reference point. At the steady-state growth conditions used, newborn cells had replicated about 50% of the chromosome. By measuring the distances of the labelled oriCs with respect to mid-cell, we found two well-separated average oriC positions in cells of newborn length. These average oriC positions moved further apart along with cell elongation. The cellular position of the ftsQAZ gene region resembled the position of oriC, although its average position was closer to mid-cell. In contrast, a single minB focus was observed at cell birth. Separated minB foci appeared towards the end of DNA replication. The average positions of oriC, ftsQAZ and minB relative to each other fitted a model in which DNA replication takes place in the cell centre and subsequent gene regions pass sequentially through this centre. We have interpreted the polarized orientation of the studied gene regions as a consequence of the mode of DNA segregation.     

E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork.

We have examined individual GATC sites throughout the E. coli genome for their kinetics of remethylation by dam methyltransferase following the passage of the chromosomal replication fork. We present evidence for three major conclusions: that oriC is a single function unit that is specifically sequestered from dam methyltransferase for a significant period of time and then released; that the dnaA promoter region is subject to sequestration analogous to that observed at oriC and thus that hemimethylation-dependent sequestration is a general phenomenon; and that each round of replication initiation triggers a transient, temporally coordinate block in both reinitiation at oriC and expression of the dnaA gene. These and other observations are all consistent with the notion that hemimethylation in these two regions acts coordinately to ensure that every origin undergoes initiation once and only once per cell cycle; other possible roles for sequestration at dnaA are also considered.