François Cornet

Macrodomain organization of the Escherichia coli chromosome

We have explored the Escherichia coli chromosome architecture by genetic dissection, using a site‐specific recombination system that reveals the spatial proximity of distant DNA sites and records interactions. By analysing the percentages of recombination between pairs of sites scattered over the chromosome, we observed that DNA interactions were restricted to within subregions of the chromosome. The results indicated an organization into a ring composed of four macrodomains and two less‐structured regions. Two of the macrodomains defined by recombination efficiency are similar to the Ter and Ori macrodomains observed by FISH. Two newly characterized macrodomains flank the Ter macrodomain and two less‐structured regions flank the Ori macrodomain. Also the interactions between sister chromatids are rare, suggesting that chromosome segregation quickly follows replication. These results reveal structural features that may be important for chromosome dynamics during the cell cycle.

KOPS: DNA motifs that control E. coli chromosome segregation by orienting the FtsK translocase

Bacterial chromosomes are organized in replichores of opposite sequence polarity. This conserved feature suggests a role in chromosome dynamics. Indeed, sequence polarity controls resolution of chromosome dimers in Escherichia coli. Chromosome dimers form by homologous recombination between sister chromosomes. They are resolved by the combined action of two tyrosine recombinases, XerC and XerD, acting at a specific chromosomal site, dif, and a DNA translocase, FtsK, which is anchored at the division septum and sorts chromosomal DNA to daughter cells. Evidences suggest that DNA motifs oriented from the replication origin towards dif provide FtsK with the necessary information to faithfully distribute chromosomal DNA to either side of the septum, thereby bringing the dif sites together at the end of this process. However, the nature of the DNA motifs acting as FtsK orienting polar sequences (KOPS) was unknown. Using genetics, bioinformatics and biochemistry, we have identified a family of DNA motifs in the E. coli chromosome with KOPS activity.

Genetic recombination and the cell cycle: what we have learned from chromosome dimers

Genetic recombination is central to DNA metabolism. It promotes sequence diversity and maintains genome integrity in all organisms. However, it can have perverse effects and profoundly influence the cell cycle. In bacteria harbouring circular chromosomes, recombination frequently has an unwanted outcome, the formation of chromosome dimers. Dimers form by homologous recombination between sister chromosomes and are eventually resolved by the action of two site‐specific recombinases, XerC and XerD, at their target site, dif, located in the replication terminus of the chromosome. Studies of the Xer system and of the modalities of dimer formation and resolution have yielded important knowledge on how both homologous and site‐specific recombination are controlled and integrated in the cell cycle. Here, we briefly review these advances and highlight the important questions they raise.

Functional polarization of the Escherichia coli chromosome terminus: the dif site acts in chromosome dimer resolution only when located between long stretches of opposite polarity

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.

FtsK activities in Xer recombination, DNA mobilization and cell division involve overlapping and separate domains of the protein.

Escherichia coli FtsK is a multifunctional protein that couples cell division and chromosome segregation. Its N‐terminal transmembrane domain (FtsKN) is essential for septum formation, whereas its C‐terminal domain (FtsKC) is required for chromosome dimer resolution by XerCD‐dif site‐specific recombination. FtsKC 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. FtsKN and FtsKC are separated by a long linker region (FtsKL) of unknown function that is highly divergent between bacterial species. Here, we analysed the in vivo effects of deletions of FtsKL and/or of FtsKC, of swaps of these domains with their Haemophilus influenzae counterparts and of a point mutation that inactivates the walker A motif of FtsKC. Phenotypic characterization of the mutants indicated a role for FtsKL in cell division. More importantly, even though Xer recombination activation and DNA mobilization both rely on the ATPase activity of FtsKC, 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.

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.

Asymmetry of Chromosome Replichores Renders the DNA Translocase Activity of FtsK Essential for Cell Division and Cell Shape Maintenance in Escherichia coli

Bacterial chromosomes are organised as two replichores of opposite polarity that coincide with the replication arms from the ori to the ter region. Here, we investigated the effects of asymmetry in replichore organisation in Escherichia coli. We show that large chromosome inversions from the terminal junction of the replichores disturb the ongoing post-replicative events, resulting in inhibition of both cell division and cell elongation. This is accompanied by alterations of the segregation pattern of loci located at the inversion endpoints, particularly of the new replichore junction. None of these defects is suppressed by restoration of termination of replication opposite oriC, indicating that they are more likely due to the asymmetry of replichore polarity than to asymmetric replication. Strikingly, DNA translocation by FtsK, which processes the terminal junction of the replichores during cell division, becomes essential in inversion-carrying strains. Inactivation of the FtsK translocation activity leads to aberrant cell morphology, strongly suggesting that it controls membrane synthesis at the division septum. Our results reveal that FtsK mediates a reciprocal control between processing of the replichore polarity junction and cell division.

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.

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.

Xer Recombination in Escherichia coli SITE-SPECIFIC DNA TOPOISOMERASE ACTIVITY OF THE XerC and XerD RECOMBINASES

Xer site-specific recombination functions in maintaining circular replicons in the monomeric state inEscherichia coli. Two recombinases of the bacteriophage λ integrase family, XerC and XerD, are required for recombination at the chromosomal site, dif, and at a range of plasmid-borne sites. Xer recombination core sites contain the 11-base pair binding sites for each recombinase separated by a 6 to 8-base pair central region. We report that both XerC and XerD act as site-specific type I topoisomerases by relaxing supercoiled plasmids containing adif site. Relaxation by either XerC or XerD occurs in the absence of the partner recombinase and requires only a single recombination core site. XerC or XerD relaxation activities are completely inhibited by the addition of the partner recombinase, providing that the DNA recognition sequence for the inhibiting partner is present.