In Escherichia coli, chromosome dimers are generated by recombination between circular sister chromosomes. Dimers are lethal unless resolved by a system that involves the XerC, XerD and FtsK proteins acting at a site (dif) in the terminus region. Resolution fails if dif is moved from its normal position. To analyse this positional requirement, dif was transplaced to a variety of positions, and deletions and inversions of portions of the dif region were constructed. Resolution occurs only when dif is located at the convergence of multiple, oppositely polarized DNA sequence elements, inferred to lie in the terminus region. These polar elements may position dif at the cell septum and be general features of chromosome organization with a role in nucleoid dynamics.

Plasmid pSC101 harbors a 28-bp sequence which is homologous to dif, the target site of the XerC/XerDdependent recombination system in Escherichia coli. Using a technique which allows very sensitive detection of plasmid loss, we show that recombination at this site, termed psi for pSC101 stabilized inheritance, causes a moderate increase in pSC101 stability. The role of the psi sequence in site-specific recombination has been explored in two other contexts. It was cloned in a derivative of plasmid pl5A and inserted into the chromosome in place of dif. In the first situation, psi activity requires accessory sequences and results in multimer resolution; in the second situation, it suppresses the effects of the dif deletion and can promote intermolecular exchanges. Thus, psi is a site whose recombinational activity depends on the context, the first in the ceridif famil known to exhibit such flexibility.

The recombination site dif is the target on the Escherichia coli chromosome of the site-specific recombinases XerC and XerD. The dif/XerC-D system plays a role during the cell cycle, probably by favoring sister chromosome monomerization or separation. A phenomenon of regional control over dif activity, also analyzed in this issue, is demonstrated here by translocation of dif to a series of loci close to the normal locus. We found that the site is physiologically active only within a narrow zone around its natural position.Competence for dif activity does not depend on the sequence of the normal dif activity zone (DAZ), because ~(diD deletions larger than the DAZ result in Dif + bacteria when dif is reinserted at the junction point. Although dif maps where replication normally terminates, termination of replication is not the elicitor. A strain with a large inversion that places dif and its surrounding region close to oriC remains Dif*, even when a Tus-mutation allows replication to terminate far away from it. Preliminary data suggest the possibility that specialized sequences separate the competent zone from the rest of the chromosome. We suspect that these sequences are members of a set of sequences involved in a polarized process of postreplicative reconstruction of the nucleoid structure. We propose that this reconstruction forces catenation links between sister chromosomes to accumulate within the DAZ, where they eventually favor recombination at d/f.[Key Words." Site-specific recombination; dif site domain of activity; E. coli chromosome organization] Received January 10, 1996; revised version accepted March 20, 1996.The

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A lambda hybrid phage (lambda Sda1), containing an 8.1 kb EcoRI DNA fragment from the Escherichia coli chromosome, was selected on the basis of its ability to suppress bacterial thermosensitivity caused by the dnaA46 mutation. We have shown that this suppression is due to a recA+-dependent amplification of the 8.1 kb fragment; consistent with this observation, cloning of the 8.1 kb fragment into a high copy number plasmid (pBR325) leads also to suppression of dnaA46. In the suppressed strains growing at high temperature, bidirectional replication starts in or near the oriC region and requires the presence of the DnaA polypeptide. These findings suggest that the overproduction of a gene product(s), encoded by the cloned 8.1 kb fragment, can restore dnaA-dependent initiation of replication at high temperature in the oriC region. Genetic mapping shows that the groES (mopB) and groEL (mopA) genes are located on the 8.1 kb suppressor fragment. Further analysis, including in vitro mutagenesis and subcloning, demonstrates that the amplification of the groES and groEL genes is both necessary and sufficient to suppress the temperature sensitive phenotype of the dnaA46 mutation.

SummaryEscherichia coli FtsK is a multifunctional protein that couples cell division and chromosome segregation. Its N-terminal transmembrane domain (FtsK N ) is essential for septum formation, whereas its Cterminal domain (FtsK C ) is required for chromosome dimer resolution by XerCD-dif site-specific recombination. FtsK C is an ATP-dependent DNA translocase. In vitro and in vivo data point to a dual role for this domain in chromosome dimer resolution (i) to directly activate recombination by XerCD-dif and (ii) to bring recombination sites together and/or to clear DNA from the closing septum. FtsK N and FtsK C are separated by a long linker region (FtsK L ) of unknown function that is highly divergent between bacterial species. Here, we analysed the in vivo effects of deletions of FtsK L and/or of FtsK C , of swaps of these domains with their Haemophilus influenzae counterparts and of a point mutation that inactivates the walker A motif of FtsK C . Phenotypic characterization of the mutants indicated a role for FtsK L in cell division. More importantly, even though Xer recombination activation and DNA mobilization both rely on the ATPase activity of FtsK C , mutants were found that can perform only one or the other of these two functions, which allowed their separation in vivo for the first time.

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.Though the architecture of the bacterial chromosome remains mysterious, the early proposal that it is established symmetrically on each replichore (31) has been supported recently by studies on the repair of the most frequent accident to befall circular chromosomes: the formation of a chromosome dimer by an odd number of recombinational exchanges between nascent chromosomes. Chromosome dimer resolution (CDR) takes place through the action of three proteins, the recombinases XerC and XerD and the septum-associated protein FtsK, on the resolution site dif (28 bp), located in the chromosome terminus (5,6,21,35). Resolution takes place at the time of division and requires septum formation (34). It also requires that dif be located within a small zone of the terminus region, the DAZ (for dif activity zone [10,22]).Failure of CDR gives rise to several phenotypes, including an increase of 50-to 100-fold in recombination in the terminus region (11,12,25,26). This terminal recombination reflects the extreme fragility of the dif region after inhibition of CDR, presumably a result of engulfment of the localized dif region by the closing septum. The recombination-stimulating events are, or culminate in, double-strand breaks, since terminal recombination is largely RecBCD dependent. Consistent with this scenario, trapping by septum of DNA joining sister nucleoids and DNA degradation near dif have been detected in CDR Ϫ mutants (18,24,30).Genetic studies of the DAZ have revealed the role played by the polarity of the regions that flank dif in generating this domain. First, the "natural" orientat...

The frequency of excisive homologous recombination has been measured at various positions along the Escherichia coli chromosome. The reporter system makes use of a X c1857 prophage integrated by homologo4s recombination within TnS or TnlO transposons already installed at known positions in the E. coli chromosome. The excision frequency per cell and per generation was determined by monitoring the evolution of the relative number of temperature-resistant (cured) bacteria as a function of the age of the cultures. Excisions, due to RecA-dependent homologous exchanges, appeared to occur more frequently in the preferential termination zone for chromosome replication. The highest frequency of excision observed is compatible with a recombination event at each replication cycle in this region. On the basis of these data, we propose a model involving homologous recombination in the final steps of bacterial chromosome replication and separation.