A dual role for the FtsK protein in Escherichia coli chromosome segregation

Abstract

FtsK is a multifunctional protein that acts in Escherichia coli cell division and chromosome segregation. Its C-terminal domain is required for XerCD-mediated recombination between dif sites that resolve chromosome dimers formed by recombination between sister chromosomes. We report the construction and analysis of a set of strains carrying different Xer recombination sites in place of dif, some of which recombine in an FtsK-independent manner. The results show that FtsK-independent Xer recombination does not support chromosome dimer resolution. Furthermore, resolution of dimers by the Cre/loxP system also requires FtsK. These findings reveal a second role for FtsK during chromosome dimer resolution in addition to XerCD activation. We propose that FtsK acts to position the dif regions, thus allowing a productive synapse between dif sites.

Fig. 1. Xer core sequences used to replace dif. wt, the wild-type dif sequence; dif, reinsertion of dif as an MluI–BglII linker. The difference core sequences are shown together with the XerC and XerD binding sites, the central region (CR) and modifications of the sequence to create BglII and MluI sites. Differences between cer3 and cer6 are shown in bold.

Fig. 2. Growth advantage of strains carrying the different Xer core sequences in place of dif over a Δ(dif) strain. Strains carrying the cer3 (closed diamonds), psi (closed triangles), cer6 (open circles) or cer (open diamonds) core sequences in place of dif were mixed with strain LN2772 [Δ(dif)58::Tc] and grown in serial culture. The relative frequency of the two strains is plotted as the Tc^S/Tc^R ratio. Closed squares, dif reinserted as an MluI–BglII linker; open squares, co-culture of strains LN2772 and FC223 (xerC::Cm); open triangles, co-culture of LN4263 (LN2772 ftsK::Cm) and LN4262 [Δ(dif)::cer3 ftsK::Cm].

Fig. 3. Recombination between plasmid-borne core sequences. Strains FX55 (ftsK wt) and FX60 (FX55 ftsK::Cm) were transformed with plasmids containing direct repetitions of the core sequences: pBH220 (dif), pBH222 (cer6) or pBH223 (cer3). Plasmid DNA was recovered after overnight growth and analysed by agarose gel electrophoresis. Positions of the substrate (S), the recombination product containing one core sequence (RP) dimers of S and RP (D), and higher multimers (M) formed by Xer recombination are indicated on the left. Note the disappearance of the forms corresponding to plasmids carrying the initial repetition of cer6 due to a higher rate of recombination at cer6 than at cer3 and dif.

Fig. 4. Phase-contrast fluorescence micrographs of strains carrying the loxP site in place of dif (DAPI staining). Top left, FC149 [LN2666 Δ(dif)::Tc::loxP–Kn–loxP]; top right, FC149/pFX71; bottom left, FC407 (FC149 ftsK::Cm); bottom right, FC407/pFX71.

Fig. 5. Viability defect due to inactivation of dif and FtsKc. Strains LN2666 (wt), FC126 [Δ(dif)2600::Gm], FC405 (ftsK::Cm) and FC318 [Δ(dif)2600::Gm ftsK::Cm] were mixed by pairs and grown in serial culture. Closed symbols, LN2666 versus each mutant strain. The relative frequency of LN2666 is plotted. Diamonds, LN2666 versus FC126; squares, LN2666 versus FC405; triangles, LN2666 versus FC318; circles, FC232 (LN2666 recA56) versus FC420 [Δ(dif) ftsK::Cm recA56]. Open symbols: mutant strain versus mutant strain. The frequency of the first strain cited is plotted. Diamonds, FC126 versus FC318; squares, FC126 versus FC405; circles, FC405 versus FC318; triangles (control), FC126 versus LN3079 [Δ(dif)2600::Tc].