Interplay between recombination, cell division and chromosome structure during chromosome dimer resolution in Escherichia coli

Chromosome dimers form in bacteria by recombination between circular chromosomes. Resolution of dimers is a highly integrated process involving recombination between dif sites catalysed by the XerCD recombinase, cell division and the integrity of the division septum-associated FtsK protein and the presence of dif inside a restricted region of the chromosome terminus, the dif activity zone (DAZ). We analyse here how these phenomena collaborate. We show that (i) both inter- and intrachromosomal recombination between dif sites are activated by their presence inside the DAZ; (ii) the DAZ-specific activation only occurs in conditions supporting the formation of chromosome dimers; (iii) overexpression of FtsK leads to a general increase in dif recombination irrespective of dif location; (iv) overexpression of FtsK does not improve the ability of dif sites inserted outside the DAZ to resolve chromosome dimers. Our results suggest that the formation of an active XerCD-FtsK-dif complex is restricted to when a dimer is present, the features of chromosome organization that determine the DAZ playing a central role in this control.     

Analysis of the terminus region of the Caulobacter crescentus chromosome and identification of the dif site

The terminus region of the Caulobacter crescentus chromosome and the dif chromosome dimer resolution site were characterized. The Caulobacter genome contains skewed sequences that abruptly switch strands at dif and may have roles in chromosome maintenance and segregation. Absence of dif or the XerCD recombinase results in a chromosome segregation defect. The Caulobacter terminus region is unusual, since it contains many essential or highly expressed genes.     

Polarization of the Escherichia coli chromosome. A view from the terminus

The E. coli chromosome replication arms are polarized by motifs such as RRNAGGGS oligomers, found preferentially on leading strands. Their skew increases regularly from the origin to dif (the site in the center of the terminus where chromosome dimer resolution occurs), to reach a value of 90% near dif. Convergent information indicates that polarization in opposite directions from the dif region controls tightly the activity of dif, probably by orienting mobilization of the terminus at cell division. Another example of polarization is the presence, in the region peripheral to the terminus, of small non-divisible zones whose inversion interferes with spatial separation of sister nucleoids. The two phenomena may contribute to the organization of the Ter macrodomain.     

Segregation of the Escherichia coli chromosome terminus

We studied the segregation of the replication terminus of the Escherichia coli chromosome by time-lapse and still photomicroscopy. The replicated termini lie together at the cell centre. They rapidly segregate away from each other immediately before cell division. At fast growth rate, the copies move progressively and quickly toward the centres of the new-born cells. At slow growth rate, the termini usually remain near the inner cell pole and migrate to the cell centre in the middle of the cell cycle. A terminus domain of about 160kb, roughly centred on the dif recombination site, segregated as a unit at cell division. Sequences outside this domain segregated before division, giving two separate foci in predivision cells. Resolution of chromosome dimers via the terminus dif site requires the XerC recombinase and an activity of the FtsK protein that is thought to align the dif sequences at the cell centre. We found that anchoring of the termini at the cell centre and proper segregation at cell division occurred normally in the absence of recombination via the XerC recombinase. Anchoring and proper segregation were, however, frequently disrupted when the C-terminal domain of FtsK was truncated.     

Site-specific recombination in the replication terminus region of Escherichia coli: functional replacement of dif.

The replication terminus region of the Escherichia coli chromosome encodes a locus, dif, that is required for normal chromosome segregation at cell division. dif is a substrate for site-specific recombination catalysed by the related chromosomally encoded recombinases XerC and XerD. It has been proposed that this recombination converts chromosome multimers formed by homologous recombination back to monomers in order that they can be segregated prior to cell division. Strains mutant in dif, xerC or xerD share a characteristic phenotype, containing a variable fraction of filamentous cells with aberrantly positioned and sized nucleoids. We show that the only DNA sequences required for wild-type dif function in the terminus region of the chromosome are contained within 33 bp known to bind XerC and XerD and that putative active site residues of the Xer recombinases are required for normal chromosome segregation. We have also shown that recombination by the loxP/Cre system of bacteriophage P1 will suppress the phenotype of a dif deletion strain when loxP is inserted in the terminus region. Suppression of the dif deletion phenotype did not occur when either dif/Xer or loxP/Cre recombination acted at other positions in the chromosome close to oriC or within lacZ, indicating that site-specific recombination must occur within the replication terminus region in order to allow normal chromosome segregation.     

Extent of the activity domain and possible roles of FtsK in the Escherichia coli chromosome terminus

Escherichia coli FtsK protein couples cell division and chromosome segregation. It is a component of the septum essential for cell division. It also acts during chromosome dimer resolution by XerCD-specific recombination at the dif site, with two distinct activities: DNA translocation oriented by skewed sequence elements and direct activation of Xer recombination. Dimer resolution requires that the skewed elements polarize in opposite directions 30-50 kb on either side of dif. This constitutes the DIF domain, approximately coincident with the region where replication terminates. The observation that the ftsK1 mutation increases recombination near dif was exploited to determine whether the chromosome region on which FtsK acts is limited to the DIF domain. A monitoring of recombination activity at multiple loci in a 350 kb region to the left of dif revealed (i) zones of differing activities unconnected to dimer resolution and (ii) a constant 10-fold increase of recombination in the 250 kb region adjacent to dif in the ftsK1 mutant. The latter effect allows definition of an FTSK domain whose total size is at least fourfold that of the DIF domain. Additional analyses revealed that FtsK activity responds to polarization in the whole FTSK domain and that displacement of the region where replication terminates preserves differences between recombination zones. Our interpretation is that translocation by FtsK occurs mostly on DNA belonging to a specifically organized domain of the chromosome, when physical links between either dimeric or still intercatenated chromosomes force this DNA to run across the septum at division.     

Dissection of a functional interaction between the DNA translocase, FtsK, and the XerD recombinase

Successful bacterial circular chromosome segregation requires that any dimeric chromosomes, which arise by crossing over during homologous recombination, are converted to monomers. Resolution of dimers to monomers requires the action of the XerCD site-specific recombinase at dif in the chromosome replication terminus region. This reaction requires the DNA translocase, FtsK(C), which activates dimer resolution by catalysing an ATP hydrolysis-dependent switch in the catalytic state of the nucleoprotein recombination complex. We show that a 62-amino-acid fragment of FtsK(C) interacts directly with the XerD C-terminus in order to stimulate the cleavage by XerD of BSN, a dif-DNA suicide substrate containing a nick in the 'bottom' strand. The resulting recombinase-DNA covalent complex can undergo strand exchange with intact duplex dif in the absence of ATP. FtsK(C)-mediated stimulation of BSN cleavage by XerD requires synaptic complex formation. Mutational impairment of the XerD-FtsK(C) interaction leads to reduction in the in vitro stimulation of BSN cleavage by XerD and a concomitant deficiency in the resolution of chromosomal dimers at dif in vivo, although other XerD functions are not affected.     

Two DNA translocases synergistically affect chromosome dimer resolution in Bacillus subtilis

In Bacillus subtilis, chromosome dimers that block complete segregation of sister chromosomes arise in about 15% of exponentially growing cells. Two dedicated recombinases, RipX and CodV, catalyze the resolution of dimers by site-specific recombination at the dif site, which is located close to the terminus region on the chromosome. We show that the two DNA translocases in B. subtilis, SftA and SpoIIIE, synergistically affect dimer resolution, presumably by positioning the dif sites in close proximity, before or after completion of cell division, respectively. Furthermore, we observed that both recombinases, RipX and CodV, assemble on the chromosome at the dif site throughout the cell cycle. The preassembly of recombinases probably ensures that dimer resolution can occur rapidly within a short time window around cell division.     

Filamentous bacteriophages of vibrios are integrated into the dif-like site of the host chromosome

The dif site is located in the replication terminus region of bacterial chromosomes, having a function of resolving dimeric chromosomes formed during replication. We demonstrate that filamentous bacteriophages of vibrios, such as f237 (Vibrio parahaemolyticus) and CTXphi (V. cholerae), are integrated into the dif-like site of host chromosome.     

dif, a recA-independent recombination site in the terminus region of the chromosome of Escherichia coli.

dif (deletion induced filamentation) is a newly identified locus that lies within the terminus region of the Escherichia coli chromosome. The Dif phenotype was characterized by a subpopulation of filamentous cells with abnormal nucleoids and induction of the SOS repair system. Interactions between dif-carrying plasmids as well as between such plasmids and the bacterial chromosome demonstrated that dif is a cis-acting, recA-independent recombination site. Filamentation continued in dif mutants in which SOS-associated division inhibitors were inoperative, which showed that induction of these inhibitors was not the primary cause of filamentation. Filamentation was not observed in dif recA or dif recBC mutants, which were unable to carry out homologous recombination. The dif site shows homology with the cer site of plasmid ColE1, which resolves plasmid multimers to monomers. It is proposed that dif functions to resolve dimeric chromosomes produced by sister chromatid exchange, and that the Dif phenotype is due to the inability of these mutants to resolve multimers prior to cell division.     

Evidence from terminal recombination gradients that FtsK uses replichore polarity to control chromosome terminus positioning at division in Escherichia coli

Chromosome dimers in Escherichia coli are resolved at the dif locus by two recombinases, XerC and XerD, and the septum-anchored FtsK protein. Chromosome dimer resolution (CDR) is subject to strong spatiotemporal control: it takes place at the time of cell division, and it requires the dif resolution site to be located at the junction between the two polarized chromosome arms or replichores. Failure of CDR results in trapping of DNA by the septum and RecABCD recombination (terminal recombination). We had proposed that dif sites of a dimer are first moved to the septum by mechanisms based on local polarity and that normally CDR then occurs as the septum closes. To determine whether FtsK plays a role in the mobilization process, as well as in the recombination reaction, we characterized terminal recombination in an ftsK mutant. The frequency of recombination at various points in the terminus region of the chromosome was measured and compared with the recombination frequency on a xerC mutant chromosome with respect to intensity, the region affected, and response to polarity distortion. The use of a prophage excision assay, which allows variation of the site of recombination and interference with local polarity, allowed us to find that cooperating FtsK-dependent and -independent processes localize dif at the septum and that DNA mobilization by FtsK is oriented by the polarity probably due to skewed sequence motifs of the mobilized material.     

All major regions of FtsK are required for resolution of chromosome dimers

Resolution of chromosome dimers, by site-specific recombination between dif sites, is carried out in Escherichia coli by XerCD recombinase in association with the FtsK protein. We show here that a variety of altered FtsK polypeptides, consisting of the N-terminal (cell division) domain alone or with deletions in the proline-glutamine-rich part of the protein, or polypeptides consisting of the C-terminal domain alone are all unable to carry out dif recombination. Alteration of the putative nucleotide-binding site also abolishes the ability of FtsK to carry out recombination between dif sites.     

KOPS-guided DNA translocation by FtsK safeguards Escherichia coli chromosome segregation

The septum-located DNA translocase, FtsK, acts to co-ordinate the late steps of Escherichia coli chromosome segregation with cell division. The FtsK γ regulatory subdomain interacts with 8 bp KOPS DNA sequences, which are oriented from the replication origin to the terminus region ( ter ) in each arm of the chromosome. This interaction directs FtsK translocation towards ter where the final chromosome unlinking by decatenation and chromosome dimer resolution occurs. Chromosome dimer resolution requires FtsK translocation along DNA and its interaction with the XerCD recombinase bound to the recombination site, dif , located within ter . The frequency of chromosome dimer formation is ∼15% per generation in wild-type cells. Here we characterize FtsK alleles that no longer recognize KOPS, yet are proficient for translocation and chromosome dimer resolution. Non-directed FtsK translocation leads to a small reduction in fitness in otherwise normal cell populations, as a consequence of ∼70% of chromosome dimers being resolved to monomers. More serious consequences arise when chromosome dimer formation is increased, or their resolution efficiency is impaired because of defects in chromosome organization and processing. For example, when Cre– loxP recombination replaces XerCD– dif recombination in dimer resolution, when functional MukBEF is absent, or when replication terminates away from ter .     

Progressive segregation of the Escherichia coli chromosome

We have followed the fate of 14 different loci around the Escherichia coli chromosome in living cells at slow growth rate using a highly efficient labelling system and automated measurements. Loci are segregated as they are replicated, but with a marked delay. Most markers segregate in a smooth temporal progression from origin to terminus. Thus, the overall pattern is one of continuous segregation during replication and is not consistent with recently published models invoking extensive sister chromosome cohesion followed by simultaneous segregation of the bulk of the chromosome. The terminus, and a region immediately clockwise from the origin, are exceptions to the overall pattern and are subjected to a more extensive delay prior to segregation. The origin region and nearby loci are replicated and segregated from the cell centre, later markers from the various positions where they lie in the nucleoid, and the terminus region from the cell centre. Segregation appears to leave one copy of each locus in place, and rapidly transport the other to the other side of the cell centre.     

Search for additional replication terminators in the Bacillus subtilis 168 chromosome.

The Bacillus subtilis 168 chromosome is known to contain at least six DNA replication terminators in the terminus region of the chromosome. By using a degenerate DNA probe for the consensus terminator sequence and low-stringency hybridization conditions, several additional minor hybridizing bands were identified. DNA corresponding to the most intense of these bands was cloned and characterized. Although localized in the terminus region, it could not bind RTP and possibly represents a degenerate terminator. A search of the SubtiList database identified an additional terminator sequence in the terminus region, near glnA. It was shown to bind RTP and to function in blocking replication fork movement in a polar manner. Its orientation conformed to the replication fork trap arrangement of the other terminators. The low-stringency hybridization experiments failed to identify any terminus region-type terminators in the region of the chromosome where postinitiation control sequences (STer sites) are known to reside. The two most likely terminators in STer site regions, in terms of sequence similarity to terminus region terminators, were identified through sequence searching. They were synthesized and were found not to bind RTP under conditions that allowed binding to terminus region terminators. Neither did they elicit fork arrest, when present in a plasmid, under stringent conditions. It is concluded that the STer site terminators, at least the first two to the left of oriC, do not have the typical consensus A+B site makeup of terminus region terminators.     

Norfloxacin-induced DNA cleavage occurs at the dif resolvase locus in Escherichia coli and is the result of interaction with topoisomerase IV

The dif locus is a site-specific recombination site located within the terminus region of the chromosome of Escherichia coli. Recombination at dif resolves circular dimer chromosomes to monomers, and this recombination requires the XerC, XerD and FtsK proteins, as well as cell division. In order to characterize other enzymes that interact at dif, we tested whether quinolone-induced cleavage occurs at this site. Quinolone drugs, such as norfloxacin, inhibit the type 2 topoisomerases, DNA gyrase and topoisomerase IV, and can cleave DNA at sites where these enzymes interact with the chromosome. Using strains in which either DNA gyrase or topoisomerase IV, or both, were resistant to norfloxacin, we determined that specific interactions between dif and topoisomerase IV caused cleavage at that site. This interaction required XerC and XerD, but did not require the C-terminal region of FtsK or cell division.     

A MatP-divisome interaction coordinates chromosome segregation with cell division in E. coli

Initiation of chromosome segregation in bacteria is achieved by proteins acting near the origin of replication. Here, we report that the precise choreography of the terminus region of the Escherichia coli chromosome is also tightly controlled. The segregation of the terminus (Ter) macrodomain (MD) involves the structuring factor MatP. We characterized that migration of the Ter MD from the new pole to mid-cell and its subsequent persistent localization at mid-cell relies on several processes. First, the replication of the Ter DNA is concomitant with its recruitment from the new pole to mid-cell in a sequential order correlated with the position on the genetic map. Second, using a strain carrying a linear chromosome with the Ter MD split in two parts, we show that replisomes are repositioned at mid-cell when replication of the Ter occurs. Third, we demonstrate that anchoring the Ter MD at mid-cell depends on the specific interaction of MatP with the division apparatus-associated protein ZapB. Our results reveal how segregation of the Ter MD is integrated in the cell-cycle control.     

Prophage lambda induces terminal recombination in Escherichia coli by inhibiting chromosome dimer resolution. An orientation-dependent cis-effect lending support to bipolarization of the terminus

A prophage lambda inserted by homologous recombination near dif, the chromosome dimer resolution site of Escherichia coli, is excised at a frequency that depends on its orientation with respect to dif. In wild-type cells, terminal hyper- (TH) recombination is prophage specific and undetectable by a test involving deletion of chromosomal segments between repeats identical to those used for prophage insertion. TH recombination is, however, detected in both excision and deletion assays when Deltadif, xerC, or ftsK mutations inhibit dimer resolution: lack of specialized resolution apparently results in recombinogenic lesions near dif. We also observed that the presence near dif of the prophage, in the orientation causing TH recombination, inhibits dif resolution activity. By its recombinogenic effect, this inhibition explains the enhanced prophage excision in wild-type cells. The primary effect of the prophage is probably an alteration of the dimer resolution regional control, which requires that dif is flanked by suitably oriented (polarized) stretches of DNA. Our model postulates that the prophage inserted near dif in the deleterious orientation disturbs chromosome polarization on the side of the site where it is integrated, because lambda DNA, like the chromosome, is polarized by sequence elements. Candidate sequences are oligomers that display skewed distributions on each oriC-dif chromosome arm and on lambda DNA.     

A cluster of cell division genes maps to the terC region of the chromosome of Escherichia coli K-12

Thirty-nine cell division mutants were isolated in Escherichia coli K-12 and were mapped in the terminus region of the chromosome, between 33.5 and 36 min. They were obtained by two different approaches involving specific mutagenesis of the terC region. The mutants could be divided into eight classes (I to VIII) based on their map position and phenotype at the restrictive temperature, and constitute a new cell division gene cluster.     

HeT-A telomere-specific retrotransposons in the centric heterochromatin of Drosophila melanogaster chromosome 3

We have isolated two yeast artificial chromosome (YAC) clones from Drosophila melanogaster that contain a small amount of dodeca satellite (a satellite DNA located in the centromeric region of chromosome 3) and sequences homologous to the telomeric retrotransposon HeT-A. Using these YACs as probes for fluorescence in situ hybridization to mitotic chromosomes, we have localized these HeT-A elements to the centric heterochromatin of chromosome 3, at region h55. The possible origin of these telomeric elements in a centromeric position is discussed. Received: 30 July 1999 / Accepted: 19 September 1999