Inversion of the metE-oriC-rpsE chromosome segment in Escherichia coli K12

We have described recently a large inversion of the Escherichia coli chromosome (designated udpPf1), including region of the chromosomal replication region (oriC). The udpPf1 inversion was induced by Tn10 transposon (metE::Tn10). It results in increased expression of the uridine phosphorylase gene (udp) which is closely linked to the metE gene. The data of conjugational and transductional experiments presented in this report demonstrate that the udpPf1 inversion covers a chromosomal segment extending over 12 min of the E. coli genetic map and including the rpsE, crp and metE::Tn5 markers. The results are presented indicating that the increased uridine phosphorylase activity is due to fusion of the udp gene to a more strong promoter located, probably, in the operon for ribosomal proteins cluster, near 73 min on the E. coli chromosome.     

Comparative genome mapping of Pseudomonas aeruginosa PAO with P. aeruginosa C, which belongs to a major clone in cystic fibrosis patients and aquatic habitats.

A physical and genetic map was constructed for Pseudomonas aeruginosa C. Mainly, two-dimensional methods were used to place 47 SpeI, 8 PacI, 5 SwaI, and 4 I-CeuI sites onto the 6.5-Mb circular chromosome. A total of 21 genes, including the rrn operons and the origin of replication, were located on the physical map. Comparison of the physical and genetic map of strain C with that of the almost 600-kb-smaller genome of P. aeruginosa reference strain PAO revealed conservation of gene order between the two strains. A large-scale mosaic structure which was due to insertions of blocks of new genetic elements which had sizes of 23 to 155 kb and contained new SpeI sites was detected in the strain C chromosome. Most of these insertions were concentrated in three locations: two are congruent with the ends of the region rich in biosynthetic genes, and the third is located in the proposed region of the replication terminus. In addition, three insertions were scattered in the region rich in biosynthetic genes. The arrangement of the rrn operons around the origin of replication was conserved in C, PAO, and nine other examined independent strains.     

Polymorphic Chromosomes Bearing the Tox2 Locus in Cochliobolus carbonum Behave as Homologs during Meiosis

The HTS1 gene in the Tox2 locus of the fungal pathogen Cochliobolus carbonum race 1 is required for synthesis of a host-selective phytotoxin and for increased virulence on susceptible genotypes of maize. The locus is present in race 1 isolates but absent from isolates of the other races, which do not produce the toxin. By pulsed-field gel electrophoresis and Southern analysis with HTS1 sequences and chromosome-specific markers, the HTS1 gene was detected on a 4-Mb chromosome in one group of isolates and on a 2.3-Mb chromosome in another group, which lacked the 4-Mb chromosome. A chromosome-specific marker from C. heterostrophus hybridized to a 2.3-Mb chromosome in non-toxin-producing isolates and in toxin-producing isolates, including those with a 4-Mb chromosome. A marker from C. carbonum hybridized to the 4-Mb chromosome, but in isolates lacking the 4-Mb chromosome, this marker hybridized to a smaller, 2.0-Mb chromosome. Thus, the Tox2 locus is on different chromosomes in different groups of race 1 isolates. Single ascospore progeny from crosses between isolates having HTS1 on different chromosomes were analyzed for toxin-producing ability, virulence, and the presence and chromosomal location of HTS1. All progeny produced HC toxin in culture, incited race 1-type lesions on susceptible maize genotypes, and contained HTS1 sequences, as determined by PCR amplification with gene-specific primers. Analysis of the chromosomal complements of several progeny indicated that they all had only one Tox2-containing chromosome. Thus, despite their differences in size, these chromosomes behave as homologs during meiosis and may have arisen by a translocation.     

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.     

The sequence requirements for a functional Escherichia coli replication origin are different for the chromosome and a minichromosome

We have developed a simple three-step method for transferring oriC mutations from plasmids to the Escherichia coli chromosome. Ten oriC mutations were used to replace the wild-type chromosomal origin of a recBCsbcB host by recombination. The mutations were subsequently transferred to a wild-type host by transduction. oriC mutants with a mutated DnaA box R1 were not obtained, suggesting that R1 is essential for chromosomal origin function. The other mutant strains showed the same growth rates, DNA contents and cell mass as wild-type cells. Mutations in the left half of oriC, in DnaA boxes M, R2 or R3 or in the Fis or IHF binding sites caused moderate asynchrony of the initiation of chromosome replication, as measured by flow cytometry. In mutants with a scrambled DnaA box R4 or with a modified distance between DnaA boxes R3 and R4, initiations were severely asynchronous. Except for oriC14 and oriC21, mutated oriCs could not, or could only poorly, support minichromosome replication, whereas most of them supported chromosome replication, showing that the classical definition of a minimal oriC is not valid for chromosome replication. We present evidence that the functionality of certain mutated oriCs is far better on the chromosome than on a minichromosome.     

The FIS protein fails to block the binding of DnaA protein to oriC, the Escherichia coli chromosomal origin.

The Escherichia coli chromosomal origin contains several bindings sites for factor for inversion stimulation (FIS), a protein originally identified to be required for DNA inversion by the Hin and Gin recombinases. The primary FIS binding site is close to two central DnaA boxes that are bound by DnaA protein to initiate chromosomal replication. Because of the close proximity of this FIS site to the two DnaA boxes, we performed in situ footprinting with 1, 10-phenanthroline-copper of complexes formed with FIS and DnaA protein that were separated by native gel electrophoresis. These studies show that the binding of FIS to the primary FIS site did not block the binding of DnaA protein to DnaA boxes R2 and R3. Also, FIS appeared to be bound more stably to oriC than DnaA protein, as deduced by its reduced rate of dissociation from a restriction fragment containing oriC . Under conditions in which FIS was stably bound to the primary FIS site, it did not inhibit oriC plasmid replication in reconstituted replication systems. Inhibition, observed only at high levels of FIS, was due to absorption by FIS binding of the negative superhelicity of the oriC plasmid that is essential for the initiation process.     

Affinity of two different regions of the chromosome to the outer membrane of Escherichia coli.

It was found that DNA associated with the outer membrane of Escherichia coli K-12 is enriched for two different regions of the chromosome, which are both on the 5.9-megadalton EcoRI fragment containing the replication origin, oriC. One region overlaps oriC, whereas the other region was found to be associated with a 1-megadalton EcoRI-BamHI fragment located within the atp operon.     

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.     

Localization and characterization of X chromosome inversion breakpoints separating Drosophila mojavensis and Drosophila arizonae

Ectopic exchange between transposable elements or other repetitive sequences along a chromosome can produce chromosomal inversions. As a result, genome sequence studies typically find sequence similarity between corresponding inversion breakpoint regions. Here, we identify and investigate the breakpoint regions of the X chromosome inversion distinguishing Drosophila mojavensis and Drosophila arizonae. We localize one inversion breakpoint to 13.7 kb and localize the other to a 1-Mb interval. Using this localization and assuming microsynteny between Drosophila melanogaster and D. arizonae, we pinpoint likely positions of the inversion breakpoints to windows of less than 3000 bp. These breakpoints define the size of the inversion to approximately 11 Mb. However, in contrast to many other studies, we fail to find significant sequence similarity between the 2 breakpoint regions. The localization of these inversion breakpoints will facilitate future genetic and molecular evolutionary studies in this species group, an emerging model system for ecological genetics.     

Megabase-scale inversion polymorphism in the wild ancestor of maize

Chromosomal inversions are thought to play a special role in local adaptation, through dramatic suppression of recombination, which favors the maintenance of locally adapted alleles. However, relatively few inversions have been characterized in population genomic data. On the basis of single-nucleotide polymorphism (SNP) genotyping across a large panel of Zea mays, we have identified an ～50-Mb region on the short arm of chromosome 1 where patterns of polymorphism are highly consistent with a polymorphic paracentric inversion that captures >700 genes. Comparison to other taxa in Zea and Tripsacum suggests that the derived, inverted state is present only in the wild Z. mays subspecies parviglumis and mexicana and is completely absent in domesticated maize. Patterns of polymorphism suggest that the inversion is ancient and geographically widespread in parviglumis. Cytological screens find little evidence for inversion loops, suggesting that inversion heterozygotes may suffer few crossover-induced fitness consequences. The inversion polymorphism shows evidence of adaptive evolution, including a strong altitudinal cline, a statistical association with environmental variables and phenotypic traits, and a skewed haplotype frequency spectrum for inverted alleles.     

Periodic formation of the oriC complex of Escherichia coli.

We examined formation of an oriC-membrane complex through the chromosome replication cycle by dot-blot hybridization using an oriC plasmid as a probe. In a wild-type culture synchronized for chromosome replication, oriC complex formation was observed periodically and transiently corresponding to the replication initiation event. Prior to initiation of replication the oriC complex was recovered in the outer membrane fraction as well as at the time of initiation of replication. Moreover, periodic formation of the oriC complex was observed even when further initiation of replication was suppressed by culturing an initiation ts mutant at the restrictive temperature. Similar periodic formation of the oriC complex was also observed when DNA elongation was inhibited by addition of nalidixic acid to the culture. However, the second periodic peak did not appear when rifampicin or chloramphenicol was added. Cells which formed the oriC complex at the restrictive temperature could immediately initiate chromosome replication when the cells were transferred to the permissive temperature. We conclude that the oriC region of Escherichia coli forms a specific complex periodically just before and at the time of initiation of chromosome replication and that oriC complex formation is a prerequisite for initiation of chromosome replication.     

The distribution and properties of RNA primed initiation sites of DNA synthesis at the replication origin of Escherichia coli chromosome.

RNA-linked DNA molecules were obtained from E. coli dnaCts cells synchronously initiating a new round of chromosome replication. The deoxynucleotides at the transition from primer RNA to DNA were 32P-labeled, and their positions were located on the nucleotide sequence of 1.4 kb genomic region (position -906 to +493) including the oriC and its leftside flanking region. In the r-strand (the counterclockwise strand), many strong transition sites were mapped in the left half portion of the oriC and a few weak sites in the left outside region. In the 1-strand (the clockwise strand), no transition sites were found inside the oriC but many weak sites were found in the left outside region. The results support the initiation mechanism in which the first leading strand synthesis starts with the r-strand counterclockwise from the oriC that is followed by the 1-strand synthesis on the displaced template strand on the left of oriC. Primer RNA molecules attached to the strong r-strand transition sites were only a few residues in length. Properties of the transition sites were discussed.     

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.     

Subcellular localization of plasmids containing the oriC region of the Escherichia coli chromosome, with or without the sopABC partitioning system

Fluorescence in situ hybridization (FISH) analysis has revealed the subcellular localization of specific chromosomal segments and plasmid molecules during the cell division cycle in Escherichia coli: the replication origin (oriC) segments on the chromosome are localized at nucleoid borders, and actively partitioning mini-F plasmid molecules are localized at the 1/4 and 3/4 positions of the cell. In contrast, mini-F plasmid molecules lacking the sopABC segment are randomly localized in cytoplasmic areas at cell poles. In this study, we analysed the subcellular localization of an oriC plasmid that contains the minimum E. coli chromosomal replication origin and its flanking regions. These oriC plasmid molecules were mainly localized in cytosolic areas at cell poles. On the other hand, oriC plasmid DNA molecules carrying the sopABC segment of F plasmid were localized at cell quarter sites, as were actively partitioning mini-F plasmid DNA molecules. Therefore, we conclude that oriC itself and its flanking regions are not sufficient for positioning the replication origin domain of the E. coli chromosome within the cell.     

High-affinity binding sites for the initiator protein DnaA on the chromosome of Escherichia coli

The initiator protein DnaA of Escherichia coli binds with unusually high affinity to five regions on the chromosome, in addition to the replication origin, oriC . Using a solid-phase DNA binding assay, in which the DNA binding C-terminal domain of DnaA is bound via a biotin tag to magnetic beads, we could fish only fragments with these six regions from different chromosomal digests. Except for oriC , these fragments contain only one or two consensus DnaA binding sites, DnaA boxes. The distribution of these high-affinity DnaA boxes on the chromosome is random.     

Regulation of the initiation of chromosomal replication in bacteria

The initiation of chromosomal replication occurs only once during the cell cycle in both prokaryotes and eukaryotes. Initiation of chromosome replication is the first and tightly controlled step of a DNA synthesis. Bacterial chromosome replication is initiated at a single origin, oriC, by the initiator protein DnaA, which specifically interacts with 9-bp non-palindromic sequences (DnaA boxes) at oriC. In Escherichia coli, a model organism used to study the mechanism of DNA replication and its regulation, the control of initiation relies on a reduction of the availability and/or activity of the two key elements, DnaA and the oriC region. This review summarizes recent research into the regulatory mechanisms of the initiation of chromosomal replication in bacteria, with emphasis on organisms other than E. coli.     

Chromosomal insertions localized around oriC affect the cell cycle in Escherichia coli

The present work reports the effects of localized insertions around the origin of Escherichia coli chromosome, oriC, on cell cycle parameters. These insertions cause an increase of the C period with an inverse correlation to the distance from oriC. In addition, Omega insertion near oriC causes an increase in the number of replication forks per chromosome, n, and Tn10 insertion causes a decrease in growth rate. We found that the same insertion positioned in another region of the chromosome, outside of oriC, has a negligible effect on the C period. Marker frequency analysis suggests a slower replication velocity along the whole chromosome. We propose that the insertions positioned at less than 2 kbp from oriC could create a structural alteration in the origin of replication that would result in a longer C period. Flow cytometry reveals that asynchrony is not associated with these alterations.     

The DnaA box R4 in the minimal oriC is dispensable for initiation of Escherichia coli chromosome replication.

We have developed a genetic system with which to replace oriC+ on the Escherichia coli chromosome with modified oriC sequences constructed on plasmids. Using this system we have demonstrated that chromosomal oriC can tolerate the insertion of a 2 kb fragment at the HindIII site between DnaA boxes R3 and R4, whereas the same insertion completely inactivates cloned oriC. We have further found that although R4 is essential for the origin activity of cloned oriC, cells carrying a deletion of R4 in chromosomal oriC are viable. These results indicate that the oriC sequence necessary for initiation of chromosome replication is different from the so-called minimal oriC that was determined with cloned oriC. Flow cytometric analyses have revealed that these oriC mutations confer the initiation asynchrony phenotype. Introduction of the R4 deletion into a fis::kan mutant, which lacks the DNA bending protein FIS, renders the mutant cells inviable.     

Distribution and organization of auxotrophic genes on the multichromosomal genome of Burkholderia multivorans ATCC 17616

The Burkholderia multivorans strain ATCC 17616 carries three circular chromosomes with sizes of 3.4, 2.5, and 0.9 Mb. To determine the distribution and organization of the amino acid biosynthetic genes on the genome of this beta-proteobacterium, various auxotrophic mutations were isolated using a Tn5 derivative that was convenient not only for the determination of its insertion site on the genome map but also for the structural analysis of the flanking regions. Analysis by pulsed-field gel electrophoresis revealed that 20 out of 23 insertion mutations were distributed on the 3.4-Mb chromosome. More detailed analysis of the his, trp, arg, and lys mutations and their flanking regions revealed the following properties of these auxotrophic genes: (i) all nine his genes were clustered on the 3.4-Mb chromosome; (ii) seven trp genes were organized within two distinct regions, i.e., a trpEGDC cluster on the 3.4-Mb chromosome and a trpFBA cluster on the 2.5-Mb chromosome; (iii) the leu gene cluster, leuCDB, was also located close to the trpFBA cluster; and (iv) lysA and argG genes were located on the 2.5-Mb chromosome, in contrast to the argH gene, which was located on the 3.4-Mb chromosome. Southern hybridization analysis, allelic exchange mutagenesis of ATCC 17616, and complementation tests demonstrated that all of the genes examined were functional and existed as a single copy within the genome. The present findings also indicated that the 2.5-Mb chromosome carried various auxotrophic genes with no structural or functional counterparts on the remaining two chromosomes.