DNA traffic in bacteria. (A) Coordination between chromosome segregation and cell division in B. subtilis and E. coli. (i) Normal vegetative cell division in E. coli or B. subtilis. When the bulk of the sister chromosomes remains at mid-cell, nucleoid occlusion factors prevent FtsZ-ring formation. Segregation of the nucleoids away from mid-cell allows the recruitment of the division machinery. (ii) Sporulation in B. subtilis. Only a third of the chromosome is trapped in the prespore compartment during the asymmetric cell division of sporulation. SpoIIIE transports the remaining two-third of the chromosome. (iii) Chromosome dimer resolution in E. coli. RecA-mediated homologous recombination during replication can lead to the formation of dimers of the circular chromosome. Replication and segregation proceed until the bulk of the sister chromosomes are distributed in each daughter cell, but the two nucleoids remain linked by DNA passing through the division septum. FtsK-dependent DNA transport brings dif sites together. FtsK subsequently activates Xer recombination to resolve the dimer into monomers. (B) Schematic representation of FtsK, SpoIIIE and the Tra proteins of two Streptomycetes conjugative plasmids. Transmembrane domains are depicted as vertical black bars and translocation modules as blue rectangles. Numbers indicate the position of amino acid residues. A grey rectangle indicates the part of FtsK_N that belongs to a ∼50 amino acid region (FtsK₅₀; aa 179–230) that increases the efficiency with which FtsK peptides carrying an intact C-terminal domain activate Xer recombination under low expression levels.

Localization and chromosome dimer resolution activity of FtsK_50LC chimaeras. (A) Schematic representation of the FtsK_50LC chimaeras as in Figure 1B. (B) In vivo pseudo-dimer resolution assay on plasmids. A scheme of the plasmid-borne substrate and product is shown on the left of the gel. dif sites are represented as black triangles. The efficiency of recombination was determined after 4 h of growth in 0.2% arabinose. (C) Localization of FtsK_50LC chimaeras fused at their C-terminal ends to YFP as in Figure 2B. (D) Growth competition between ftsA^* ftsK⁻ and ftsA^* ftsK⁻ Δdif cells ectopically producing the FtsK_50LC chimaeras as in Figure 2C.

Fully efficient chromosome dimer resolution by FtsK chimaeras. (A) Growth competition between ftsK_LC⁻ cells ectopically expressing wild-type FtsK or FtsK chimaeras from a low copy vector under the ftsK promoter. f represents the growth disadvantage of the strain producing the FtsK chimaera compared to the strain producing wild-type FtsK. (B) Scheme of the chromosomal dif-cassette (left drawing) and of the recombinant product (right drawing) that is used to monitor the efficiency with which FtsK chimaeras induce Xer recombination at the normal dif locus on the chromosome. dif sites are represented by black triangles. Arrows indicate the positions of the primers used in the qPCR assay. Primers X/Z serve to quantify the relative number of chromosomes in which the DNA cassette between the two repeated dif sites is excised (right drawing). To this aim, genomic DNA needs to be digested with HindIII (dashed line, H3) to prevent contamination by larger products encompassing the cassette. Primers X/Y serve to quantify the relative number of chromosomes that still harbour the dif-cassette. (C) Chromosomal dif-cassette excision on ectopic production of full-length FtsK or FtsK chimaeras. The % of cassette excision was monitored by qPCR at different times after the induction of the production of the ftsK alleles. The actual data points and best-fit lines from a typical experiment are shown on the left. The slopes of the best-fit lines provide a good estimate of the rate of cassette excision as a function of time, which allowed us to calculate the relative efficiency of cassette excision of each chimaera when compared with full-length FtsK. The mean relative efficiencies are indicated on the right (results from three independent experiments for ZipA∷FtsK_LC and FtsW∷FtsK_LC and from two independent experiments for FtsK_LC).

Localization and chromosome dimer resolution activity of FtsK_LC chimaeras. (A) Schematic view of septum assembly. The protein icons are ordered from left to right according to the commonly accepted assembly sequence. OM, outer membrane; IM, inner membrane. (B) Schematic representation of the FtsK_LC chimaeras as in Figure 1B. (C) Localization of FtsK_LC chimaeras fused at their C-terminal ends to YFP and of full-length FtsK fused at its N-terminal end to GFP in ftsK⁺ cells. A similar localization pattern was observed in ftsA* ftsK⁻ and in ftsK_LC⁻ cells (data not shown). (C) Pictures of ftsK⁺ cells are shown because of their simpler cell division phenotype. Scale bars (white bars), 1 μm. (D) Growth competition between ftsA^* ftsK⁻ and ftsA^* ftsK⁻ Δdif cells. The graph of a typical result is shown. f, mean and standard deviation of the growth advantage given by the ectopic production of each FtsK_LC chimaera (result from three independent experiments).

Role of linker in chromosome dimer resolution. (A) Scheme of the FtsK peptides tested for their chromosome dimer resolution ability. Numbers on the bottom refer to the amino acid position residues delimiting the different FtsK_L deletions. The range of the deletion size is indicated on the left. f, mean and standard deviation of the growth advantage given by the ectopic production of each FtsK_LC chimaera (result from three independent experiments). The relative efficiency of chromosome dimer resolution is indicated as follows: >70% (+++); 35–70% (++); <35% (+); 0% (–). ND, not determined. (B) In vivo pseudo-dimer resolution assay on plasmids for the FtsW∷FtsK_LΔ500C chimaera as in Figure 3B. (C) Localization of the YFP derivative of FtsW∷FtsK_LΔ500C as in Figure 2C.

Schematic of DNA transport by FtsK. (A, B) Limiting steps for chromosome dimer resolution in the hypothesis in which FtsK transports DNA through a pore formed by its N-terminal domain: transfer of the last segment of the circular double-stranded chromosome (A) and formation of a recombination synapse between dif sites carried by a dimer when they are positioned on either side of a closed septum at cell division (B). (C) The assembly of several FtsK complexes in an incompletely formed septum ensures synapsis of dif sites carried by a dimer and the transfer of circular DNA molecules. (D) The inertia of chromosomes drives the C-terminal domains of active FtsK molecules towards the side of the division plan from which DNA is pumped until the maximal extension of the linker is reached. To avoid clutter, only one of the six linkers attaching FtsK hexamers to the internal membrane of the cell is drawn.