Y-recombinases are also widespread and may play a role in various DNA transactions (5,26). Homologs of XerC and XerD, together with a potential *dif* site opposite the origin of replication, are found in most proteobacteria and firmicutes (27–29). Other families of bacteria use alternative systems to resolve chromosome dimers (28,29). In streptococci/lactococci, a family of firmicutes, the dimer resolution system consists of a single Y-recombinase, XerS, which is responsible for recombination at a specific recombination site, *dif*<sub>SL</sub> (Figure 1A) (28). The XerS/*dif*<sub>SL</sub> system is the only single-recombinase Xer system functionally studied to date. However, it is certainly not the only such system. It has recently been reported that a subgroup of ε-proteobacteria harbour only one conserved Y-recombinase, XerH, together with a potential *dif* site resembling *dif*<sub>SL</sub> and located within the putative terminus regions of these bacteria (29). In evolutionary terms, XerH is only distantly related to XerCD and XerS, consistent with the acquisition of Xer systems on several occasions during evolution (29).

The *Lactococcus lactis* XerS/*dif*<sub>SL</sub> system resolves chromosome dimers in an FtsK<sub>C</sub>-dependent manner if it is used to replace the XerCD/*dif* system in *E. coli* (28). This observation seems to go against the highly accurate control of chromosome dimer resolution in *E. coli*, which involves specific interactions between partners, and raises questions about the possible control of dimer resolution by a single recombinase. In this study, we investigated the molecular mechanism of XerS/*dif*<sub>SL</sub> recombination and its control by FtsK. We show that XerS binds and recombinases *dif*<sub>SL</sub> sites *in vitro*. Both binding and strand exchange were asymmetric. We showed, using *in vivo* assays, that the FtsK-dependent coupling of chromosome segregation with cell division is conserved in the FtsK/Xer system of *L. lactis*. However, the mechanism by which recombination was activated differed from that in the *E. coli* system. FtsKγ can activate XerCD/*dif* recombination on its own, whereas the translocation motor is also required to activate XerS/*dif*<sub>SL</sub> recombination. These findings have implications for the control of recombination in the two Xer systems.

## EXPERIMENTAL PROCEDURES

### Strains and plasmids

Strains used were derived from *E. coli* K12 strain LN2666 [W1485 F- *leu* thyA *thi* deoB or *C* supE *rpsL* (StR)], referred as the wt strain (30). Unless indicated, recombinant DNA was constructed in transgenesis vectors of the pFC13 series by standard cloning techniques, and inserted into the chromosome as previously described (30,31). The *dif-lacI-dif* and *dif*<sub>SL</sub>-*lacI-dif*<sub>SL</sub> cassettes were inserted in place of the Δ(*dif*)<sub>58</sub> deletion (30). Three 5'-GGGCAGGG KOPS motifs separated by 6-bp random sequences (5'-TT CATT and 5'-TAGCAT) were eventually inserted in a non-permissive orientation, 10 bp from the XerD binding site of the *dif* site on one side of the *dif-lacI-dif* cassette or at the equivalent position in the *dif*<sub>SL</sub>-*lacI-dif*<sub>SL</sub> cassette. The Δ(*lacI*) deletion removes bp -37 to 1075 of the *lacI* gene. In the Δ(*ftsK*<sub>C</sub>):Tc allele, the residues from

position 814 to the end of FtsK are deleted. The Δ(*ftsK*γ)-Cm, *ftsK*<sub>KOPSblind</sub>-Cm and Δ(*xerC*)::Gm alleles have been described elsewhere (32). Resistance gene-tagged alleles were transferred by P1 transduction. For protein production and purification, *xerS* and the *E. coli* and *L. lactis* *ftsK*γ subdomains (corresponding to the sequence from residue 1253 of *E. coli* FtsK and 682 of *L. lactis* FtsK to the ends of these proteins) were inserted into pFSKB3X (GTP Technology, France) to obtain *his-flag-gene* constructs. We then mutated *xerS*, using mutagenic oligonucleotides, to obtain the *xerS*<sup>Y341F</sup> allele. For *in vivo* expression of the recombinases, genes were inserted into a pGB2 derivative carrying the *araC-ARAp* expression cassette, yielding plasmids pFC241 (pXerC), pCP56 (pXerS) and pFC250 (pXerC-XerD). For FtsKγ and FtsK<sub>C</sub> production, the pBAD18 derivatives, pFX150 (pFtsK<sub>C</sub>) (7), pCL379 (p-γE.coli) and pCL378 (p-γL.lactis) were used. For subsequent *in vitro* analysis, a synthetic minimal *dif*<sub>SL</sub> site, 5'ATCTTTCCGAAAAAC TGTAATTTTCTTGACA and its variants lacking the right-half site, 5'ATCTTTCCGAAAAACTGTAAGGC TACGTCAT, or the left-half site, 5'CTGAGCTAGTCA CACTGTAATTTTCTTGACA, were inserted into pGhost9 (33).

### Purification of XerS

For XerS and XerS<sup>Y341F</sup> purification, pCL255 (XerS) and pCL360 (XerS<sup>Y341F</sup>) were transferred to *E. coli* strain BL21 (DE3; Novagen). The resulting strains were grown in L broth at 42°C, to an OD<sub>600</sub> of 0.6. We then added IPTG (0.1 mM) to the medium and incubated the culture at 25°C for 3 h. Cells were recovered by centrifugation, resuspended in buffer [50 mM phosphate buffer pH 8, 500 mM NaCl, 10 mM imidazole, 10% glycerol, 1 mg/ml lysosyme, 230 µg/ml RNaseA and EDTA-free protease inhibitor cocktail (Roche), sonicated and the lysate was cleared by centrifugation. His-FLAG-tagged XerS and XerS<sup>Y341F</sup> were purified on nickel resin (1 ml His-trap HP, GE Healthcare) followed by heparin (1 ml High-Trap HP, GE Healthcare) and gel filtration (HiLoad 16/60 Superdex 200, GE Healthcare) columns (Supplementary Figure S1). Purified recombinases were concentrated with an ICON concentrator (Thermoscientific) and stored at -80°C in buffer containing 40 mM Hepes (pH 7.7), 400 mM KCl, 1 mM DTT, 0.5 mM EDTA and 10% glycerol.

### In vitro experiments

For electrophoretic mobility shift assays (EMSAs), 142-bp fragments carrying the site of recombination at their centre were amplified by PCR and purified by gel electrophoresis. These substrates were 5' end-labelled with [γ-³²P] ATP and T4 DNA polynucleotide kinase. Binding reactions were carried out in a buffer containing 25 mM Hepes (pH 7.7), 50 mM KCl, 0.25 mM EDTA, 0.5 mM DTT, 10 µg/ml BSA, 10 mM MgCl₂ and 10% glycerol, in the presence of 10 000 c.p.m. of labelled DNA (<1 nM), 1 µg of poly(dI-dC) and the indicated protein concentrations. The reactions were incubated at 30°C for 30 min and analysed by electrophoresis in 5% polyacrylamide native
gels run in TGE. Gels were dried and analysed with a Fuji
PhosphorImager.

For DNAse1 footprinting analysis, various amounts of XerS were mixed with 226-bp *dif*<sub>SL</sub>-carrying DNA fragments labelled with ³²P at the 5' end of either the top or the bottom strand (BS). Reactions were performed as for EMSA. After 30 min of incubation at 30°C, we added 7 mM MgCl₂ and 3 mM CaCl₂, and partial digestion of the DNA was initiated by adding 1 µl of an empirically determined dilution (typically 10⁻³) of a DNase I stock solution. The mixture was incubated for 2 min at 30°C and the reaction was stopped by adding 1 µl EDTA (0.5 M). The DNA was then precipitated in ethanol, re-suspended in 6 µl of loading buffer and separated by electrophoresis in an 8% polyacrylamide denaturing sequencing gel run in TBE. A sequencing reaction was carried out with the Thermo Sequenase Cycle Sequencing Kit (USB) and was run in parallel, as a size marker. Gels were dried and analysed with a PhosphorImager.

For recombination experiments, the HJ substrate (Figure 3) was constructed by annealing four oligonucleotides:

(I) 5'-CTGCCGTGATCACGCTGAACGCGTTTTAGC ATCTTTCCGAAAAACTGTAATTTTCTTGACAA TTGGAGGCCTAACGCCTAAAGCGGCCGCCTA GTCC;

(II) 5'-GGACTAGGCGGCCGCTTTAGGCGTTAGGC CTCCAATTGTCAAGAAAATTACAGTTTTTCGG AAAGATCGACCGTTGCCGGATCCGCTGC;

(III) 5'-GGATTCGAGATCTCAGGATGTCTCCAATT GTCAAGAAAATTACAGTTTTTCGGAAAGATG CTAAAACGCGTTCAGCGTGATCACGGCAG;

(IV) 5'-GCAGCGGATCCGGCAACGGTCGATCTTTT CCGAAAAACTGTAATTTTCTTGACAATTGGAG ACATCCTGAGATCTCGAATCC.

The indicated oligonucleotide (20 pmol) was 5' end-labelled with [γ-³²P] ATP and T4 DNA polynucleotide kinase and was mixed with the other, unlabelled oligonucleotides (30 pmol each). The oligonucleotides were annealed by heating to 100°C and slow cooling. The HJ-containing molecules were purified by PAGE in a 5% polyacrylamide gel, recovered from excised gel slices by incubation in elution buffer [10 mM Tris-HCl (pH 8), 1 mM EDTA, 0.2% SDS, 0.3 M NaCl, 1 mg/ml glycogen], precipitated in ethanol and re-suspended in deionized water. Reactions were performed as described earlier for EMSA (except that the reaction buffer contained 5 mM spermidine) and were stopped by adding 1% SDS and 1 mM EDTA. The samples were then treated with proteinase K (1 h at 37°C) and the DNA was analysed by PAGE in 5% polyacrylamide native gels in (Tris/glycin/EDTA) TGE or in 8% polyacrylamide denaturing gels (containing 7 M urea) in 8% TBE.

Co-culture experiments

Co-culture experiments were conducted as previously
described (34). Briefly, the strains to be analysed were
mixed with the parental strains [either wt or Δ(dif):dif<sub>SL</sub>
pXerS strains]. In the case of strains containing the

pFtsK<sub>C</sub> plasmid, 0.025% arabinose was added during
the whole course of the experiment. The mixture of
strains was grown in serial cultures, subcultured by
dilution every 10 generations and plated every 20 gener-
ations on appropriate medium to determine the relative
frequencies of the two strains. Frequencies of unresolved
dimers were calculated from the slope of strain ratio
plotted against generation (34). The means of at least
three independent measurements, with SDs, are presented.

XerCD/dif and XerS/dif<sub>SL</sub> *in vivo* recombination assay

Strains carrying the Δ(lacI) and *xerC::Gm* mutations, either the *dif-lacI-dif* or the *difSL-lacI-difSL* cassette in place of the *dif* site and the indicated *ftsK* alleles and plasmids were grown in L broth, rendered competent and transformed with pXerC or pXerS. Transformants were plated on L agar containing 20 µg/ml spectinomycin and grown overnight at 37°C. Five independent transformants were re-suspended in L broth plus spectinomycin and, in some cases, arabinose (0.025% for FtsK<sub>C</sub> production and 0.1% for FtsK<sub>γ</sub> production), grown 5 h, diluted and plated on L broth plus X-gal (40 µg/ml). This entire procedure corresponds to ~20 generations before plating on L agar plus Xgal. The ratio of dark blue to total colonies was used to calculate the frequency of *lacI* loss per cell, per generation. The mean and SD of five independent measurements are shown in the figures.

Western-blot analysis

Strains were grown in the assay conditions, harvested at an OD<sub>600</sub> of 0.1 and resuspended in sample buffer (1% SDS, 10% glycerol, 0.1 M DTT, 0.2% bromophenol blue, 50 mM Tris-HCl, pH 6.8). Samples were boiled for 5 min. We used ~0.04 OD units for each sample. Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes (Hybond-C Extra, GE Healthcare). Membranes were then incubated with mouse anti-FLAG primary antibodies (Sigma), followed by goat anti-mouse IgG peroxidase-linked secondary antibodies (Sigma), as indicated by manufacturer, and antibody binding was detected with an ECL kit (Thermo Scientific) and a BioImager (LAS-4000 FUJIFILM).

RESULTS

Asymmetric binding of XerS to *dif*<sub>SL</sub>

The potential *dif*<sub>SL</sub> site has been defined as a minimal 31-bp site, on the basis of comparative genomics studies of streptococci and *in vivo* functional assays of recombination activity (Figure 1A) (28). This site contains two imperfect inversely repeated sequences of different sizes that are potential binding sites for XerS (the right and left-half sites in Figure 1), separated by a CR. For studies of XerS binding to *dif*<sub>SL</sub>, we constructed a *his-flag-xerS* allele. We produced the protein encoded by this allele (herein referred to as XerS), purified it (Experimental procedures section, Supplementary Figure S1) and used it in EMSA experiments with various *dif*<sub>SL</sub>-containing DNA fragments. We found that the previously defined minimal
$dif_{SL}$ site bound XerS poorly, with efficient binding requiring additional nucleotides flanking the right arm (data not shown). Nucleotides other than those naturally flanking $dif_{SL}$ could give efficient binding, demonstrating that these nucleotides are not specific, accounting for their lack of conservation among $dif_{SL}$ sites from different streptococci [Figure 1A; (28)].

At low-XerS concentration, a first XerS/$dif_{SL}$ complex (C1) is detected, which is transformed into a second lower mobility complex (C2) with increasing XerS concentration (Figure 2A). Considered together with data from other Y recombinase systems, these data suggest that the C1 complex consists of a single XerS monomer bound to one half site of $dif_{SL}$, with the binding of a second monomer to the second half site giving rise to the C2 complex. We then studied the location of XerS binding in DNasel footprinting experiments (Figure 2B and Supplementary Figure S2). In these experiments, XerS protected both inverted repeats, together with the CR and few base pairs adjacent to the right arm. Thus, the non-specific base pairs next to the right-half site required for efficient binding are also protected from DNasel cleavage. XerS binding also increased sensitivity to DNasel cleavage on either side of $dif_{SL}$, suggesting that the DNA is distorted at these positions, which thus probably correspond to the first unbound bases on either side of the XerS/$dif_{SL}$ complex (Figure 2B and Supplementary Figure S2). Thus, the $dif_{SL}$ site is longer than previously thought and the left and right XerS binding sites are of similar lengths.

The left and right-half sites of $dif_{SL}$ are not perfect inverted repeats (Figure 1A). We therefore studied XerS binding to the two half sites separately. We did this by using $dif_{SL}$ variants in which one of the half sites had been mutated. Both half sites formed a complex equivalent to the C1 complex (Figure 2C and D) in these experiments, consistent with our hypothesis that this complex consists of a single XerS monomer bound to one half site. XerS bound the left arm with a much higher affinity than the right arm (compare Figure 2C and D). This suggests that XerS binds preferentially to the left arm and co-operatively to the right arm of $dif_{SL}$. At high-XerS concentration, a second complex is detected with DNA carrying the low affinity right-half site (C3 in Figure 2D). XerS binding to the right-half site may thus promote the binding of a second XerS monomer to non-specific adjacent sequences. However, this complex appeared to be different from the C2 complex, indicating that the left arm DNA is required for the formation of a C2 complex in the correct conformation.

## Strand preference in XerS/$dif_{SL}$ recombination

As XerS belongs to the Y-recombinase family, the recombination it mediates should involve a HJ-containing intermediate, produced by the exchange of a first pair of strands by a pair of recombinases and with product resolution subsequently achieved by the exchange of the second pair of strands by the other pair of recombinases [reviewed in (5); Figure 1B]. The catalytic state of pairs of recombinases thus switches from active to inactive and

vice versa, during the HJ step of the reaction, making this intermediate a target of choice for control of the reaction. This led us to assay XerS-mediated cleavage and strand exchange with HJ-containing substrates. We constructed a HJ-containing substrate that mimicked classical intermediates of Y recombinase-driven recombination (35–37) (Figure 3A). The incubation of this HJ substrate with XerS yielded linear products of the expected size following cleavage by both pairs of recombinases (B1 and T1 in Figure 3B). Thus, XerS is able to cleave both the $dif_{SL}$ strands. Covalent complexes between XerS and the DNA were detected in the absence of proteinase K treatment (Supplementary Figure S3). These complexes are intermediate between the cleavage and the ligation steps during strand exchange (Figure 1B). The small quantity of these covalent complexes suggested that religation occurs efficiently to give rise to exchanged strands. We assessed strand transfer directly, by analysing HJ substrates by electrophoresis in denaturing gels after incubation with XerS (Figure 3C and Supplementary Figure S3). The predicted recombinant strands were readily detected in these experiments, showing that XerS does indeed transfer and religate DNA strands. The two pairs of strands are not exchanged equally efficiently, with the BSs clearly being exchanged preferentially (Figure 3B). The two pairs of recombinases therefore do not have equivalent activities at this step in the recombination reaction, demonstrating a marked asymmetry of cleavage-competent XerS/$dif_{SL}$ complexes.

Alignment of the amino acid sequence of XerS with those of other known Y recombinases led to the identification of conserved residues involved in catalysis, including Y341, which was identified as the residue likely to be responsible for DNA cleavage (Supplementary Figure S4A). We used mutagenesis to replace this residue by a phenylalanine residue ('Experimental procedures' section). The resulting protein was purified and its $dif_{SL}$ binding and cleavage were assessed. XerS$^{Y341F}$ bound $dif_{SL}$, albeit slightly less efficiently than wild type (wt) XerS (Supplementary Figure S4B). As predicted, XerS$^{Y341F}$ was unable to cleave the HJ-containing substrate (Figure 3B), demonstrating the involvement of Y341 in catalysis.

Taken together, these results show that XerS binds $dif_{SL}$ and exchanges its DNA strands *in vitro*. Both activities were found to be highly asymmetric: binding to the left-half site was more efficient than binding to the right-half site and a preferential exchange of the BSs was observed. As other known bacterial Y recombinases cleave DNA *in cis* (Figure 1B) (5), we would expect the BSs of $dif_{SL}$ to be cleaved by recombinases bound to its right-half site, which interacts poorly with XerS.

## Translocation by septum-borne FtsK controls dimer resolution by XerS/$dif_{SL}$

We studied the control of chromosome dimer resolution by XerS/$dif_{SL}$, using *E. coli* strains harbouring $dif_{SL}$ in place of the *dif* site and measuring the capacity of these strains to resolve dimers, by co-culture with a parental reference strain ['Experimental procedures' section; (34)].
<figure><img src="image_107.png" /><figcaption>**Figure 2.** Asymmetric binding of XerS to *dif*<sub>SL</sub>. (A) Titration of a 142-bp DNA fragment containing *dif*<sub>SL</sub> by increasing concentrations of XerS in an EMSA experiment. The DNA substrate is shown at the top. XerS concentrations are given in nano molar and the positions of the free DNA probes and the C1, and C2 XerS/DNA complexes are indicated. The asterisk indicates the 5'-labelled strand. (B) DNasel footprinting of the top strand (TS) of *dif*<sub>SL</sub>. Experiments were carried out with increasing XerS concentrations, as described in the 'Experimental procedures' section. A, C, T and G, ladder sequence of the *dif*<sub>SL</sub>-containing substrate; (-) lane, no XerS; Lanes 1, 2, 3 and 4: reactions contained 12.5, 25, 50 and 100 nM XerS, respectively. The region protected from cleavage is indicated by the black bars and positions of increased cleavage by arrows. (C and D) Same experiments as in (A), with substrates containing mutated right- and left-half sites, respectively. (E) Summary of the data for the *dif*<sub>SL</sub> sequence. The footprint of the BS is shown in Supplementary Figure S2. Black bars, protected regions; Arrows, positions hypersensitive to cleavage.</figcaption></figure>

The Δ(dif) :: dif<sub>SL</sub> strain producing XerS from a plasmid (pXerS) displayed a clear growth advantage over the dif<sup>+</sup> Δ(xerC) strain, which is unable to resolve dimers (Figure 4B and C). On the other hand, this strain

displayed no viability difference with the wt strain,
which resolves dimers by XerCD/dif recombination
(Figure 4B and C). Thus XerS/dif<sub>SL</sub> is as efficient as
XerCD/dif for the resolution of chromosome dimers in
<figure><img src="image_108.png" /><figcaption>**Figure 3.** Strand exchange by XerS. (A) Left: diagram of the HJ substrate with the labelled strand indicated by asterisk. The left and right *dif*<sub>SL</sub> half sites are indicated by pale grey and dark grey, respectively. Right: resolution products (T1, T2 and B1, B2; with their respective sizes indicated) obtained by exchange of either the top or bottom pairs of strands (defined in Figure 2). Labelled strands are indicated by asterisk. (B) PAGE analysis of the recombination products with positions of the substrate (HJ), B1 and T1 products indicated. (–) lane, no XerS; XerS and XerS<sup>Y341F</sup> lanes, reactions contained 200 mM of the protein indicated. (C) Analysis of the individual DNA strands by denaturing PAGE. Positions of the labelled strands are indicated. (AGCT) lanes, size ladder ('Experimental procedures' section). (–) lane, no XerS; (+) lane: reaction contained 200 nM XerS.</figcaption></figure>

*E. coli.* Mutant alleles of *ftsK* (Figure 4A) were then transferred to the Δ(*dif*)::*dif*<sub>SL</sub> pXerS strain. The deletion of *FtsK*<sub>C</sub> or the mutation of its ATP hydrolysis motif both inactivate dimer resolution in *E. coli* (38,39). These mutations also inactivated dimer resolution by the *XerS/dif*<sub>SL</sub> system, demonstrating a dependence of the dimer resolution activity of *XerS/dif*<sub>SL</sub> on *FtsK*<sub>C</sub> and its translocation activity. We then produced the C-terminal domain of *FtsK* from a plasmid in Δ(*ftsK*<sub>C</sub>) strains, in expression conditions that support *XerCD/dif* recombination (see below and 'Experimental procedures' section). This form of *FtsK* that lacks the N-terminal domain and is thus not bound to the division septum does not support dimers resolution by the *XerCD/dif* system (10,40). Although coculture experiments with strains containing p*FtsK*<sub>C</sub> showed important variations and high standard deviations (SD), it was clear that *FtsK*<sub>C</sub> did not support significant rates of dimer resolution (Figure 4B). Thus, dimer resolution by the *XerS/dif*<sub>SL</sub> systems requires a septum-borne *FtsK*.

We assayed recombination directly, by constructing strains carrying two directly repeated *dif*<sub>SL</sub> sites flanking the *lacI* gene (Figure 5A; 'Experimental procedures' section). This *dif*<sub>SL*-lacI-*dif*<sub>SL</sub> cassette was inserted in place of the *dif* site, in a strain carrying a deletion of *lacI*. This made possible the accurate assessment of recombination events, evaluated by scoring the number of blue colonies after transformation with pXerS. As previously reported, XerS/*dif*<sub>SL</sub> recombined at high frequency (~12% per generation) when inserted at the *dif* position, and recombination was found to be dependent on *FtsK*<sub>C</sub> (Figure 5B) (28). As previously shown for XerCD/*dif* (10), inactivation of the ATPase activity of *FtsK* inactivated XerS/*dif*<sub>SL</sub> recombination, showing that *FtsK* translocation is a prerequisite for the activation of XerS/*dif*<sub>SL</sub> recombination. The production of *FtsK*<sub>C</sub> in the Δ(*ftsK*<sub>C</sub>) strains significantly increased the frequency of XerS/*dif*<sub>SL</sub> recombination (Figure 5B). Thus, septum-independent *FtsK*<sub>C</sub> is sufficient to induce XerS/*dif*<sub>SL</sub> recombination, but only septum-tethered *FtsK*<sub>C</sub> can induce chromosome dimer resolution.

## FtsKγ activates XerS/dif<sub>SL</sub> recombination via a local effect

The data reported above suggest that recombination is controlled in similar ways in the *L. lactis* and *E. coli* Xer systems: both the XerS/*dif*<sub>SL</sub> and XerCD/*dif* systems resolve chromosome dimers in an *FtsK*<sub>C</sub>-dependent manner and, in both cases, recombination can be induced by septum-independent *FtsK*<sub>C</sub>, but dimer resolution can only occur if *FtsK*<sub>C</sub> is tethered to the septum [Figures 4 and 5; (10,38,40,41)]. This suggests that the *L. lactis* *FtsK* homologue, *FtsK*<sub>LI</sub>, exerts the same regulatory activity as the *E. coli* *FtsK*. We therefore replaced the C-terminal domain of *E. coli* *FtsK* by its homologue in *FtsK*<sub>LI</sub> ('Experimental Procedures' section) and measured its capacity to induce XerS/*dif*<sub>SL</sub> and XerCD/*dif* recombination. We used strains carrying equivalent *dif*<sub>SL</sub>-lacI-*dif*<sub>SL</sub> or *dif*-lacI-*dif* cassettes and producing the cognate recombinase from plasmids ('Experimental procedures' section). The resulting *ftsK*<sub>CLI</sub> chimera supported high frequencies of recombination by both the XerS/*dif*<sub>SL</sub> and XerCD/*dif* systems (Figure 6B). Thus, either the activities of *FtsK*<sub>C</sub> are conserved between these two bacteria or some of these activities are dispensable for normal levels of recombination.

In *E. coli*, the *FtsK*γ subdomain is responsible for the recognition of both KOPS and XerD. The level of identity of the *E. coli* and *L. lactis* *FtsK*γ subdomains (41% over 61 residues; Figure 6A) suggests that these activities may be conserved in *FtsK*<sub>LI</sub>. However, the presence of several changes in important residues is not consistent with this hypothesis. The TEKRKA motif of *FtsK*, which appears to interact with XerD (20,39,42), is only weakly conserved, and changes in the H2 and H3 helices of the W-helix motif may have made it possible for *FtsK*<sub>LI</sub> to recognize DNA motifs other than the *E. coli* KOPS motif (Figure 6A). We compared the roles of *FtsK*γ activities in XerS/*dif*<sub>SL</sub> and XerCD/*dif* recombination, by first using a Δ(*ftsK*γ) allele. As previously reported for the XerCD/*dif* system (32), the
<figure><img src="image_109.png" /><figcaption>**Figure 4.** Resolution of chromosome dimers by XerS/dif<sub>SL</sub>. (A) Diagram of the FtsK mutants used. Top line, wt *E. coli* FtsK with the three domains and the C-terminal subdomains indicated (α,β,γ). Black bars represent the A and B Walker-type motifs. Co-ordinates at the top are in amino acid. The grey bar labelled (A) indicates the mutation of the FtsK<sub>ATP</sub> mutant. The FtsK<sub>C</sub> domain was produced from a plasmid (pFtsK<sub>C</sub>; 'Experimental procedures' section) under the control of the pBAD promoter and was used in Δ(*ftsK*<sub>C</sub>) strains in the presence of 0.025% arabinose. (B) Left: typical co-culture experiments. A Δ(*dif*)::dif<sub>SL</sub> strain carrying the pXerS plasmid was co-cultured with strains carrying either the *dif* site [Δ(*xerC*) and wt strains] or the *dif*<sub>SL</sub> site in place of *dif*, the pXerS plasmid and the indicated *ftsK* allele [Δ(*ftsK*<sub>C</sub>), *ftsK*<sub>ATP</sub>, and Δ(*ftsK*<sub>C</sub>) pFtsK<sub>C</sub> strains]. Right: frequencies of unresolved dimers in the indicated strains, calculated from experiments in B (34). The mean results of at least three independent experiments with standard deviations are shown.</figcaption></figure>

<figure><img src="image_110.png" /><figcaption>**Figure 5.** XerS/dif<sub>SL</sub> recombination depends on the translocation activity of FtsK<sub>C</sub>. (A) The recombination reporter cassettes used consist of two directly repeated dif<sub>SL</sub>, separated by the *lacI* gene, inserted at the natural position of *dif*. Recombination deletes *lacI*, giving rise to blue colonies on indicator medium. An example of assessments made by plating is shown. (B) Recombination frequencies in strains carrying the dif<sub>SL</sub>-lacI-dif<sub>SL</sub> cassette and the indicated *ftsK* alleles ('Experimental procedures' section). Proteins encoded by the Δ(*ftsK*<sub>C</sub>) and *ftsK*<sub>ATP</sub>- mutations and the pFtsK<sub>C</sub> plasmid are shown in Figure 4A. Strains containing pFtsK<sub>C</sub> were grown in absence of inducer (NI) or in presence (I) of 0.025% arabinose.</figcaption></figure>
<figure><img src="image_111.png" /><figcaption>**Figure 6.** FtsK must reach *dif*<sub>SL</sub> to induce recombination. (A) Alignment of the FtsKγ subdomains of *E. coli* (Ec) and *L. lactis* (Ll). Structural features of the Ec FtsKγ are indicated: the H1-3 helices and the wing of the w-helix DNA-binding domain. Identical residues are shown in bold and residues for which mutation leads to the KOPS-blind phenotype are indicated by stars. The regions thought to interact with XerD and the KOPS motif are indicated. The co-ordinate of the last residue aligned here is given to the right. (B) Recombination frequencies in strain carrying either a *dif-lacI-dif* (left, XerCD/dif) or a *dif*<sub>SL</sub>-*lacI-dif*<sub>SL</sub> (right, XerS/dif<sub>SL</sub>) recombination cassette, the pXerC or pXerS plasmids, respectively, and the indicated *ftsK* alleles and/or insertion of non-permissive KOPS motifs close to one of the recombination sites (3KOPSi; 'Experimental procedures' section). The Δ(*ftsK*<sub>C</sub>):/*ftsK*<sub>CLl</sub> allele carries the whole C-terminal domain of *L. lactis* FtsK in place of the *E. coli* domain ('Experimental procedures' section; Figure 4A). The *ftsK*<sub>KOPSblind</sub> allele carries mutations of the three residues indicated by stars in (A).</figcaption></figure>

deletion of FtsKγ had the same effect on XerS/dif<sub>SL</sub> recom-
bination as deletion of the entire FtsK<sub>C</sub> (Figure 6B). Thus,
the translocation activity of FtsK is not sufficient to induce
XerS/dif<sub>SL</sub> recombination, with at least one of the FtsKγ
activities being required. We then inactivated the KOPS
recognition activity of FtsK, using the *ftsK*<sub>KOPSblind</sub> allele
(Figure 6A) (16,32). This almost completely inactivated
XerS/dif<sub>SL</sub> recombination, demonstrating a requirement
for FtsK to translocate to *dif*<sub>SL</sub> to activate recombination.
It follows that, as the FtsK<sub>CLl</sub> chimera supports recombin-
ation, FtsK<sub>LI</sub> either recognizes the *E. coli* KOPSs or a
motif with an orientation sufficiently biased in the *ter*
region of the *E. coli* chromosome to direct translocation
towards the *dif* site. We investigated whether FtsK needed
to reach the XerS/dif<sub>SL</sub> complex to induce recombination,
by inserting three KOPS motifs (5'-GGGCAGGG motifs
separated by 6-bp random sequences; 'Experimental pro-
cedures' section), 10 bp from the recombination cassettes
in a non-permissive orientation, thereby preventing FtsK
from reaching one side of the cassette (12). This significant-
ly reduced XerS/dif<sub>SL</sub> recombination in *ftsK*<sub>wt</sub> conditions
but had no effect in *ftsK*<sub>KOPSblind</sub> conditions (Figure 6B).
The same effects were observed in equivalent strains
carrying the *dif-lacI-dif* cassette (Figure 6B). We
conclude that FtsK must reach the XerS/dif<sub>SL</sub> complex
for the induction of XerS/dif<sub>SL</sub> recombination.

A differential role for FtsKγ

The data presented above and previous reports suggest
that FtsK must deliver FtsKγ to the recombination

complex to induce recombination through a direct inter-
action with the recombinases. However, for XerS/dif<sub>SL</sub>,
this appears to be inconsistent with the poor conservation
of the TEKRKA motif in FtsK<sub>LI</sub> (Figure 6A).
Furthermore, the XerD residues identified as potentially
interacting with FtsKγ are not conserved in XerS
(Figure 7A). To investigate the mechanisms underlying
the activation of recombination, we first attempted to set
up in vitro recombination using purified FtsK<sub>50C</sub> (7) and
XerS proteins. We repeatedly failed to observe XerS/dif<sub>SL</sub>
recombination in conditions supporting XerCD/dif re-
combination (data not shown), which further suggested
a difference in the mechanism of XerCD/dif and XerS/
dif<sub>SL</sub> recombination induction. We then assayed the
FtsKγ-mediated activation of recombination in vivo. To
this aim, we assessed the effect of *ftsKγ* expression in
strains with deletions of either *ftsK<sub>C</sub>* or *ftsKγ* carrying
the recombination cassettes. Control experiments in
these mutant strains showed that XerS supported higher
levels of FtsK<sub>C</sub>- and FtsKγ-independent recombination
than XerCD (1 and 0.01%, respectively; Figure 7B). The
*E. coli* and *L. lactis* FtsKγ genes were cloned and fused to
a FLAG tag for subsequent western analysis and plasmids
producing one or other of these FtsKγ proteins were
introduced into the Δ(*ftsKγ*) and Δ(*ftsK<sub>C</sub>*) strains.
Western analysis showed that *E. coli* FtsKγ and *L. lactis*
FtsKγ were produced to similar levels in the assay condi-
tions used (Figure 7B; 'Experimental procedures' section).

*Escherichia coli* FtsKγ supported recombination by the
*E. coli* XerCD/dif system in both Δ(*ftsK*<sub>C</sub>) and Δ(*ftsK*γ)
<figure><img src="image_112.png" /><figcaption>**Figure 7.** FtsKγ induces XerCD/dif but not XerS/dif<sub>SL</sub> recombination. (A) Alignment of the FtsK interaction region of XerD with its corresponding region in XerS. Residues inferred to interact with FtsK are indicated and shown in bold typeface [R1300A, E1303A, E1306A; (21)]. The co-ordinate of the last residue is given to the right. (B) Western-blot analysis of FtsKγ production. Cells were collected from the indicated strains during the course of the experiments shown in (C). His-Flag-tagged *E. coli* and *L. lactis* FtsKγ were detected with an anti-FLAG antibody ('Experimental procedures' section). M, Purified *E. coli* His-FLAG-FtsKγ, with its size indicated. The faint bands that migrate faster than the FtsKγ peptides are background detection in the migration front of the gel. (C) Recombination frequencies in strains carrying either a *dif*-lact-dif (left, XerCD/dif) or a *dif*<sub>SL</sub>-lact-dif<sub>SL</sub> (right, XerS/dif<sub>SL</sub>) recombination cassette and the indicated ftsK alleles and FtsKγ-coding plasmid. Recombination was induced by transformation with the pXerC plasmid (all strains in the XerCD/dif panel except strains labelled pXerCD for which the pXerC-XerD plasmid was used; 'Experimental procedures' section), or the pXerS plasmids (XerS/dif<sub>SL</sub> panel). NI, absence of inducer; I, FtsKγ production was induced by adding 0.1% arabinose. The dotted lines and grey zones indicate the FtsK<sub>C</sub>-independent recombination background for the two systems. In both cases, the recombination background in the absence of the cognate recombinase is below 10<sup>-3</sup> (data not shown).</figcaption></figure>

strains (Figure 7C). Thus, the translocation activity of FtsK is not required for the induction of recombination and it is not essential for FtsKγ to be tethered to FtsK<sub>C</sub> for the induction of recombination. In contrast, *E. coli* FtsKγ did not support recombination by the XerS/dif<sub>SL</sub> system (Figure 7C). The *L. lactis* FtsKγ did not support recombination by either of the Xer systems (Figure 7C). In these experiments, XerS is produced from a plasmid, while XerCD/dif recombination is analysed in strains containing normal levels of XerD, making it possible that the difference observed between the two Xer systems is due to a titration of FtsKγ by excess XerS ('Experimental procedures' section). We ruled out this possibility by co-producing XerD and XerC from the same plasmid and in the same conditions as XerC or XerS alone ('Experimental procedures' section). This yielded equivalent levels of FtsKγ-induced XerCD/dif recombination as production of XerC alone (Figure 7B), showing that FtsKγ titration by excess recombinase does not explains the observed effects. Taken together, these data strongly suggest that there is no FtsKγ-XerS interaction functionally equivalent to the FtsKγ-XerD interaction in the *L. lactis* FtsK-XerS system.

DISCUSSION

We have shown in this study that the control of chromo-
some dimer resolution and its coupling with cell division
are mostly conserved between evolutionary remote

FtsK-Xer systems involving either one or two recombinases. Indeed, despite this important difference, the *E. coli* XerCD/dif and the *L. lactis* XerS/dif<sub>SL</sub> systems have several important characteristics in common, in terms of their recombination mechanism and its control by the FtsK translocase. The mode of binding of XerS to dif<sub>SL</sub> is similar to the mode of binding of XerCD to dif. Both recombinase/DNA complexes are highly asymmetric, with one of the two half sites bound with higher affinity [Figure 2; (43)]. Similarly, cleavage and strand exchange are also asymmetric in these two systems. XerS preferentially exchanged the BSs of dif<sub>SL</sub> on HJ-substrates, which, assuming XerS cleaves DNA in cis, would be cleaved by the monomer bound with weaker affinity to the right-half site (Figure 3). Similarly, XerC displays less efficient binding but more efficient strand exchange than XerD, with dif-containing HJ substrates (35,44). The control of recombination by FtsK is also similar in the two systems. Both Xer systems require FtsK<sub>C</sub> translocation activity for efficient recombination, and a physical tethering of FtsK<sub>C</sub> to the division septum for the resolution of chromosome dimers [Figures 4 and 5; (10,38,41)]. The XerCD/dif system also appears to be faithfully controlled by the *L. lactis* FtsK<sub>C</sub> and the XerS/dif<sub>SL</sub> system is controlled by *E. coli* FtsK in assays *in vivo* in *E. coli* [Figure 6; (28)]. These results unambiguously show that the mode of chromosome dimer resolution and its coupling with cell division by FtsK described in *E. coli* can operate in Xer systems including only one recombinase. We therefore
cannot account for the selection of two-recombinase
systems in most bacteria due to a need for two enzymes
for the control of recombination. Importantly, these
controls also apply to non rod-shaped bacteria from
phyla other than Proteobacteria. These findings conflict
with those obtained for *Bacillus subtilis*, which, like *L.
lactis*, belongs to the Firmicute phylum. *Bacillus subtilis*
encodes a 'classical' XerCD/dif chromosome resolution
system, the activity of which has been reported to be in-
dependent of both the FtsK homologues present in this
bacterium, SpoIIIE and SftA (formerly YtpT) (45).
However, it has recently been reported that the inactiva-
tion of SftA results in a high frequency of septum closure
over the nucleoids and that this effect depends on RecA
(24). This suggests that SftA may control chromosome
dimer resolution in *B. subtilis*, although it may not
directly control XerCD/dif recombination.

A key conserved feature of the XerCD/dif and XerS/
dif<sub>SL</sub> systems is that efficient recombination, in addition to
efficient dimer resolution, requires an activation step de-
pendent on FtsK<sub>C</sub>. Efficient XerCD/dif recombination has
been shown to occur at a time corresponding to sept-
ation—long after the replication of *dif*—and to depend
on the activities of FtsZ and FtsI (46,47). Thus, the
observed dependence on FtsK<sub>C</sub> certainly ensures that re-
combination occurs only at the very end of the segregation
process, when FtsK reaches the zone of converging KOPS
containing the *dif* site, thereby preventing undesirable re-
combination events creating dimers from monomers or
producing other harmful rearrangements. This notion is
supported by the data obtained with strains in which
chromosome dimers are resolved by the Cre/loxP system
(48). In such strains, recombination between loxP sites
inserted in place of *dif* does not depend on FtsK, but
the resolution of dimers requires FtsK<sub>C</sub> activity (38).
The inactivation of FtsK<sub>C</sub> leads to a viability defect sig-
nificantly greater than that observed in strains carrying the
XerCD/dif or XerS/dif<sub>SL</sub> system [(32), compare with
Figure 4B]. This strongly suggests that Cre/loxP recom-
bination readily creates dimers in the absence of FtsK<sub>C</sub>
activity. We thus assume that dependence on FtsK<sub>C</sub>
prevents harmful recombination events, including dimer
formation.

The requirement for an activation step in XerCD/dif
and XerS/dif<sub>SL</sub> recombination indicates that the recom-
bination synaptic complexes do not assemble efficiently
in the absence of FtsK<sub>C</sub> or that they do not give rise to
productive recombination. A feature clearly linked to this
control is the asymmetry of the recombination complexes.
In Y-recombinase systems, asymmetry is required for the
correct assembly of the synaptic complexes [reviewed in
(5)]. The use of two different recombinases in the
XerCD/dif system clearly adds an additional level of
asymmetry. This certainly contributes to the weak
activity of the XerCD/dif complex in the absence of
FtsK<sub>C</sub> (Figure 7). In this view, the conformation that ac-
tivates XerD catalysis is highly improbable at both the
synapsis and HJ intermediate steps, so productive recom-
bination occurs only when imposed by FtsK. A similar
control mechanism seems to operate in the XerS/dif<sub>SL</sub>
system, although may be less stringent, hence the higher

recombination activity in the absence of FtsK<sub>C</sub> of the
XerS/dif<sub>SL</sub> compared to the XerCD/dif system. Notably,
this difference is not due to different levels of recombinase
production (Figure 7C). Thus, the use of two recombin-
ases, incorporating an additional level of asymmetry, may
allow tighter control of recombination than is possible
with a single recombinase. The control exerted over re-
combination events may also involve a differencial
control of individual strand exchanges. In the XerCD/dif
system, the exchange of a first pair of strands can occur in
the absence of FtsK<sub>C</sub>, producing the HJ intermediate
in vitro and in vivo (10). It may be essential to keep
XerCD/dif complexes active for XerC-mediated strand
exchange, for the integration of foreign DNA by XerCD
recombination. This is the case during integration of the
Vibrio cholerae CTX prophage, in which XerC catalyses
the formation of an integration intermediate that is not
further processed by XerD-mediated catalysis (9). This
mechanism may be general to proteobacteria, in which
an increasing number of mobile elements have been
shown to integrate into the host dif site (49–51). Tight
control of the exchange of the two pairs of strands may
not be possible in single-recombinase Xer systems. Two
recombinase systems may therefore have been selected due
to their role in horizontal gene transfer. Consistent with
this idea, no mobile genetic element integrated into the
streptococcal or lactococcal dif<sub>SL</sub> sites has been reported
to date.

The mechanism by which FtsK activates recombination
remains unclear. Our data show that FtsKγ is sufficient to
induce XerCD/dif recombination independently of FtsK<sub>C</sub>
(Figure 7). In these conditions, FtsKγ interacts with XerD
and induces the XerD-mediated exchange of either the
first or the second pair of strands. Whatever the mechan-
ism involved in these recombination events, it contrasts
with the lack of recombination observed with the
ftsK<sub>ATP</sub> allele (39), which encodes an intact FtsKγ
subdomain and may thus induce recombination in the
absence of translocation. In addition, an FtsK<sub>CATP</sub>
mutant protein produced in conditions equivalent to
those used here for FtsKγ production did not induce
XerCD/dif recombination (10). These data suggest that,
when tethered to FtsKαβ, FtsKγ cannot induce recombin-
ation in the absence of translocation. FtsKγ may thus be
inactivated by an interaction with FtsKαβ, which is
released during translocation, or when FtsK reaches the
XerCD/dif complex. *Lactococcus lactis* FtsKγ cannot
induce recombination by itself, even through the XerS/
dif<sub>SL</sub> system. There is therefore no interaction equivalent
to the *E. coli* FtsKγ-XerD interaction in the *L. lactis*
system (Figure 7). Nevertheless, the FtsK<sub>CL1</sub> chimera
induces high levels of recombination by both systems,
strongly suggesting that the specific FtsKγ-XerD inter-
action is also dispensable for the induction of XerCD/dif
recombination (Figure 6). This finding contrasts with a
previous report that an FtsK chimera carrying the
C-terminal domain of *Haemophilus influenzae* FtsK is de-
ficient in XerD activation, resulting in a large deficit in
XerCD/dif recombination in *E. coli* (39). However,
further analysis of this FtsK allele suggests that this
chimeric protein is also deficient for the recognition of
KOPS motifs, which certainly accounts for the recombination deficiency observed in strains carrying this chimera (C. Pages and F. Cornet, unpublished data).

So, what is the likely mechanism for recombination in-
duction common to *E. coli* and *L. lactis*? The observation
that the insertion of non-permissive KOPS motifs next to
a *dif*<sub>SL</sub> site strongly inhibits XerS/*dif*<sub>SL</sub> recombination
strongly suggests that FtsK must come into close
contact with the XerS/*dif*<sub>SL</sub> complex to activate recombin-
ation (Figure 6). We thus hypothesize that recombination
induction involves a special activity of the FstKαβ trans-
location motor on reaching the recombination complex
(18). FtsK stops translocating at this point. This cessation
of translocation is not dependent on FtsKγ and is not
highly sequence-specific, since translocation also stops
with Cre/*loxP* and XerCD/*dif* complexes (18). It is
accompanied by a particular interaction with bases imme-
diately flanking the *dif* site and, thus, also those flanking
the *dif*<sub>SL</sub> site (19). This suggests a special action of FtsK
on the DNA at this step, which may be the major factor
indicating an active conformation to the XerCD/*dif* or
XerS/*dif*<sub>SL</sub> complexes. The specific interaction of FtsKγ
with XerD would then be an additional but dispensable
control, potentially helping to stabilize the active conform-
ation and/or inducing XerD catalysis.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

ACKNOWLEDGEMENTS

We thank Jean-Yves Bouet for technical assistance and
critical reading of the article and the members of the
Cornet and Ritzenthaler groups for assistance and
helpful discussions.

FUNDING

Cnetre National de la Recherche Scientifique; University
Paul Sabatier; Fondation pour la Recherche Medicale
(FRM, to S.N.); Agence Nationale de la Recherche
(ANR; contract BLAN06-2 134012). Funding for open
access charge: ANR (contract BLAN06-2 134012).

*Conflict of interest statement.* None declared.

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50. Gonzalez,M., Lichtensteiger,C., Caughlan,R. and Vimr,E. (2002) Conserved filamentous prophage in Escherichia coli O18:K1:H7 and Yersinia pestis biovar orientalis. *J. Bacteriol.*, **184**, 6050-6055.

51. Hamilton,H., Domínguez,N., Schwartz,K., Hackett,K. and Dillard,J. (2005) Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system. *Mol. Microbiol.*, **55**, 1704-1721.
<figure><img src="image_113.png" /><figcaption>**Figure S1:** Purification of XerS.</figcaption></figure>

SDS-PAGE analysis of XerS purification. Strain BL21(DE3) containing plasmid pCL255 was grown in L broth at 42°C to OD₆₀₀=0.6, then incubated at RT after addition of IPTG (0.1 mM) for 3 hours. Cells were recovered by centrifugation, resuspended in lysis buffer and sonicated. The resulting lysate was cleared by centrifugation. His-FLAG-tagged XerS was purified on nickel resin (1 ml His-trap HP, GE Healthcare, N) followed by heparin (1 ml High-trap HP, GE Healthcare, H) and gel filtration (High-load 16/60 Superdex 200, GE Healthcare, GF) columns. The stars indicate truncated XerS forms that are detected in western-blot analysis using anti-FLAG antibodies. The gel was stained with coomassie blue.
<figure><img src="image_114.png" /><figcaption>**Figure S2:** DNAseI footprinting of the bottom strand (BS) of *dif*<sub>SL</sub>. Experiments were done at increasing XerS concentrations as described in Experimental procedures. A, C, T and G: ladder sequence of the *dif*<sub>SL</sub>-containing substrate. - : no XerS. Lanes 1, 2, 3 and 4: 12.5, 25, 50 and 100nM of XerS, respectively. The regions protected from cleavage with the left-right orientation of the site are indicated by black bars and positions of increased cleavage by arrows.</figcaption></figure>
<figure><img src="image_115.png" /></figure>

<figure><img src="image_116.png" /><figcaption>**Figure S3:** Strand exchange by XerS.</figcaption></figure>

A) Left: diagram of the HJ substrate with the strand numbered. The left (pale grey) and right (dark grey) *dif*<sub>SL</sub> half sites are indicated. Right: resolution products (T1, T2 and B1, B2; with their respective sizes indicated) obtained by exchange of either the top (1 and 4) or bottom (2 and 3) pairs of strands (see also Figure 2). B) Analysis of the individual DNA strands by denaturing PAGE. Positions of the labelled strands are indicated. (AGCT) lanes: size ladder (Experimental procedures). HJ2, 3 and 4: HJ substrates labelled on strands 2, 3 or 4, respectively; (-) lane: no XerS; (+) lane: reaction contained 200 nM XerS. C) PAGE analysis of the recombination products. Left: reaction were treated with proteinase. Right: the proteinase treatment was omitted. The positions of the substrate (HJ), B1, B2, T1 and T2 products indicated. CC indicated the position of XerS-DNA covalent complexes that are present only in the absence of proteinase treatment. (-) lane: no XerS; (+) lanes: reactions contained 200 mM XerS.
A

<table><tr><td>L. lactis XerS</td><td>297</td><td>RVTPHKLRTHTLATRLYAQTNSQVLVSNQLGHASTQVTDLYTHIINEEQKNALDNL----</td><td>357</td></tr><tr><td>E. coli XerD</td><td>234</td><td>KLSPHVLRHAFATHLLNHGADLRVVQMLLGHSDLSTTQIYTHVATERLRQLHQQHPRA</td><td>298</td></tr><tr><td>H. influenzae XerD</td><td>231</td><td>ALSPHVLRHAFATHLVNHGADLRVVQMLLGHTDLSTTQIYTHVAKERLKRLHERFHPRG</td><td>297</td></tr><tr><td>P1 Cre</td><td>279</td><td>AWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD</td><td>343</td></tr></table>

catalytic tyrosine

B

<figure><img src="image_117.png" /><figcaption>**Figure S4:** Binding of XerS<sup>Y342F</sup> to *dif*<sub>SL</sub>-containing DNA.</figcaption></figure>

A) Alignment of tyrosine recombinases revealing the position of residues involved in catalysis in XerS (shown in red with the catalytic tyrosine indicated). B) The substrates are cartooned on the top of the gel. XerS<sup>Y342F</sup> concentrations are given in nM and the positions of the free DNA probes and the C1 and C2 XerS/DNA complexes are indicated.
<figure><img src="image_118.png" /><figcaption>**Fig. 12.** Comparaison de la distribution et de l'orientation des motifs KOPS/SRS chez *E. coli*, *B. subtilis*, et *L. lactis*.</figcaption></figure>

<figure><img src="image_119.png" /><figcaption>**Fig. 13.** Représentation schématique des protéines chimères $3\gamma_{EC}$ et $3\gamma_{LI}$. EC, *E. coli*; LI, *L. lactis*.</figcaption></figure>
Données complémentaires.

Les sites KOPS canoniques ne sont pas conservés chez L. lactis.

Chez *E. coli*, l'orientation de la translocation du chromosome par FtsK est dictée par les motifs KOPS (FtsK Orienting Polar Sequences), une famille d'octamères de séquence 5'-GGGNAGGG-3' dont l'orientation est fortement biaisée suivant l'axe oriC/dif (Bigot *et al.*, 2005). Ces motifs sont parmi les plus surreprésentés et biaisés en orientation chez les γ-protéobactéries (Hendrickson & Lawrence, 2006) et certaines α-protéobactéries, comme *Rhizobium leguminosarum* (Young *et al.*, 2006). Chez *B. subtilis*, l'espèce modèle des Firmicutes, un octamère légèrement différent, appelé SRS pour SpolIIIE-Recognition Sequence, a été mis en évidence (Ptacin *et al.*, 2008). Il a été montré que la spécificité de reconnaissance entre les motifs KOPS et SRS, de séquence 5'-GAGAAGGG-3', était strictement liée aux sous-domaines γ de FtsK et de SpolIIIE (Ptacin *et al.*, 2008). Cependant, les KOPS et SRS ne diffèrent que de 2 nucléotides, ce qui suggère l'existence d'une forte pression de sélection pour une conservation du motif responsable de la directionnalité de la translocation du chromosome bactérien par les translocases à ADN double brin de la famille FtsK/SpolIIIE.

Chez *L. lactis* et les autres streptocoques, ni les KOPS ni les SRS ne présentent les caractéristiques attendues pour orienter la translocation de FtsK<sub>SL</sub> (Fig. 12). En effet, ils sont peu présents et, de ce fait, n'apparaissent pas biaisés sur le chromosome. Ces données suggèrent fortement que s'il existe un motif orientant FtsK<sub>SL</sub> chez *L. lactis*, il est différent des mots décrits chez les autres espèces bactériennes. Afin de valider cette hypothèse et déterminer le motif guidant la translocation de FtsK chez *L. lactis*, nous avons utilisé une combinaison d'approches in silico, in vitro et in vivo.

1.1) Utilisation d'une protéine chimère.

Sachant que le sous-domaine γ seul d'E. coli possède une très faible affinité pour les KOPS
(Sivanathan et al., 2006) et qu'un motif KOPS est reconnu par trois sous-domaines γ (Löwe et al.,
2008), nous avons décidé de construire une protéine chimère constituée de trois sous-domaines γ
reliés par des « linkers » riches en glycine (Fig. 13). La séquence « linker » choisie est celle qui a
permis de créer des trimères du domaine C-terminal de la protéine FtsK d'E. coli (Crozat et al., 2010).
Nous avons ainsi construit deux gènes synthétiques, l'un codant la protéine 3γLI constituée des sous-
domaines γ de L. lactis, et l'autre codant la protéine 3γEC composée des sous-domaines γ d'E. coli. Les
protéines possèdent à leur extrémité N-ter une étiquette « 6xhis » et une étiquette « Flag » pour
faciliter leur purification et leur détection. Ces deux protéines ne multimérisent pas spontanément,
et la protéine 3γEC est capable d'activer, in vivo chez E. coli, la recombinaison XerCD/difEC. Ce résultat
est comparable à celui obtenu avec le sous-domaine γEC seul (Nolivos et al., 2010), indiquant que la
<figure><img src="image_120.png" /><figcaption>**Fig. 14.** La protéine 3γ<sub>Ec</sub> se fixe efficacement sur un ADN contenant trois KOPS chevauchants.</figcaption></figure>

Expérience de retards sur gel réalisée avec un ADN contenant trois KOPS chevauchants, dont la séquence est indiquée en dessous du gel. Les traits en dessous de la séquence indiquent les motifs KOPS. L'ADN a été incubé soit en absence (-), soit en présence de concentrations croissantes en protéines 3γ<sub>Ec</sub> (triangle blanc) ou 3γ<sub>L1</sub> (triangle rouge) et en absence (-) ou en présence (+) d'ADN compétiteur (polydIdC). L'ADN libre est indiqué, ainsi que les complexes nucléoprotéiques formés (C1 et C2).
conformation adoptée par la protéine chimère maintient cette activité. Ce test ne permet pas de
spéculer sur l'activité de $3\gamma_{LI}$ puisque, comme le sous-domaine $\gamma_{LI}$ seul, il n'active ni la recombinaison
à dif, ni la recombinaison à $dif_{SL}$ (Nolivos et al., 2010). Cependant, les résultats obtenus avec les
protéines d'E. coli nous confortent dans l'idée que les domaines y reliés en trimères par une
séquence flexible ont conservé toutes leurs activités.

## 1.2) La protéine $3\gamma_{EC}$ d'E. coli se fixent efficacement sur des KOPS chevauchants.

L'effet des KOPS sur la translocation de FtsK est cumulatif, c'est-à-dire qu'un motif a peu d'effet alors qu'une succession de KOPS à un effet important sur la translocation (Bigot et al., 2005). Pour cette raison, nous avons tout d'abord regardé par des expériences de retard sur gel (EMSA) la fixation des protéines $3\gamma_{Ec}$ d'E. coli sur des ADN portant 3 KOPS chevauchants (Fig. 14). En présence des protéines $3\gamma_{Ec}$, une grande partie de l'ADN est retardé et on observe la formation d'un complexe nucléoprotéique C1. Les expériences ont été réalisées en absence (-) et en présence (+) d'ADN compétiteur (polydldC), ce qui ne change pas le profil observé, démontrant que les complexes observés correspondent à des interactions spécifiques. Néanmoins, nous n'observons pas une seule bande nette, ce qui indique que les complexes formés sont peu stables car ils se dissocient pendant l'électrophorèse. L'instabilité des complexes est en accord avec le rôle biologique de cette interaction, puisqu'une interaction trop forte entre la protéine FtsK et le KOPS irait à l'encontre de son activité de translocation. On remarque également que, à très forte concentration, la protéine $3\gamma_{LI}$ forme un complexe (C2) en absence et en présence d'ADN compétiteur. Le complexe semble migrer à la même distance que celui formé par la protéine d'E. coli, ce qui suggère que la stœchiométrie des deux complexes est identique. Ce résultat nous indique donc que la protéine $3\gamma_{LI}$ reconnaît spécifiquement cet ADN et probablement le motif KOPS. Cependant, bien que le complexe formé semble stable, très peu d'ADN est retardé, démontrant que l'affinité de la protéine pour cette séquence est faible. Associé au fait que le motif GGGNAGGG n'est pas surreprésenté et peu biaisé sur le génome (Fig. 12a), ce résultat suggère que les KOPS d'E. coli ne sont pas de bons candidats pour orienter la translocation de FtsK de L. lactis.

## 1.3) La protéine $3\gamma_{LI}$ de L. lactis ne reconnaît pas les AIMS.

Le terme d'AIMS (Architecture IMparting Sequences) définit des octamères nucléotidiques surreprésentées et biaisées suivant l'axe oriC-dif, et supposées maintenir la structure polarisée du chromosome (Hendrickson & Lawrence, 2006). Leur fonction n'est pas définie, mais la distribution biaisée de ces motifs sur le chromosome suggère qu'ils sont impliqués dans des processus biologiques qui utilisent la polarité de l'origine au terminus de réplication. Chez L. lactis, sur les quatre AIMS déterminés (Fig. 15), aucun ne correspond aux KOPS ou aux SRS, en accord avec leur
<figure><img src="image_121.png" /><figcaption>**Fig. 15.** Distribution et orientation des motifs AIMS sur le génome de *L. lactis*. A chaque représentation est associée l'expérience de gel retard correspondante. **A)** AAGAAGAT; **B)** GAATTAGA; **C)** TGGAGAAA; **D)** TGGAGGAA; **E)** localisation des AIMS sur une région de 10 kb centrée sur *dif*<sub>SL</sub>.</figcaption></figure>
faible représentation sur le génome (Fig. 12). Nous avons vérifié expérimentalement qu'aucun des
ces motifs n'était reconnu par le sous-domaine y de FtsK<sub>SL</sub>. Nous avons testé la fixation des deux
protéines 3y sur des ADN portants une succession de trois motifs AIMS (Fig. 15). En absence de
compétiteur (poly[dI-dC]), la fixation des protéines est dépendante de la séquence. Ainsi, le 3y<sub>Ec</sub>
interagit avec les ADN portant les motifs AAGAAGAT (Fig. 15A), TGGAGAAA (Fig. 15C), et TGGAGGAA
(Fig. 15D), et le 3y<sub>Ll</sub> avec l'ADN AAGAAGAT (Fig. 15A) et de manière très faible avec l'ADN TGGAGGAA
(Fig. 15D). Il apparaît donc qu'on puisse détecter un niveau faible d'interactions spécifiques à l'ADN
pour les protéines 3y. La présence de poly[dI-dC], au lieu de révéler la spécificité d'interaction,
semble dissocier les complexes formés. Néanmoins, pour les quatre séquences testées, les quantités
d'ADN retardé sont bien inférieures à la fixation de la protéine 3y<sub>Ec</sub> sur 3 KOPS chevauchants (Fig.
14). De plus, les motifs AAGAAGAT et TGGAGGAA reconnus par le 3y<sub>Ll</sub> sont présents une seule fois à
proximité du site *dif*<sub>SL</sub>, et dans une orientation inverse à celle attendue pour une translocation du
chromosome vers *dif*<sub>SL</sub> (Fig. 15E). Il semble que chez *L. lactis* aucun des quatre motifs AIMS ne joue le
rôle de KOPS.

1.4) Recherche de nouveaux octamères nucléotidiques biaisés et surreprésentés sur le chromosome
de L. lactis.

Les résultats précédents ne permettaient d’envisager que deux possibilités : soit il n’existe pas
de site de type KOPS chez les lactocoques, soit les motifs sont très différents. Nous avons donc
engagé une collaboration avec Meriem El Karoui et Fabrice Touzain (INRA Jouy-en-Josas) afin de
définir les candidats potentiels parmi les octamères biaisés sur le chromosome de *L. lactis*, en
imposant différents paramètres comme la surreprésentation et le biais d’orientation (Halpern *et al.*,
2007). Pour définir les bornes de cette région, nous nous sommes basés sur les données obtenues
chez *E. coli*. En effet, le domaine d’activité de FtsK chez *E. coli* est d’environ 350 kb (Corre & Louarn,
2005), ce qui correspond à 7 % du génome. Nous avons donc considéré chez *L. lactis* une région de
7 % du génome (environ 150 kb) centrée sur *dif*<sub>SL</sub>, la taille de cette région est très inférieure au
« domaine Ter » précédemment déterminé (voir **article 3**). L’analyse a été réalisée sur le génome des
deux sous-espèces de *L. lactis*, celui de la souche IL1403 pour la sous-espèce *lactis* et celui de la
souche MG1363 pour la sous-espèce *cremoris* (Fig. 16A). Cette étude a permis de proposer deux
candidats, les motifs AGAAGAAG et AAGAAGAA, pour le génome de la souche MG1363, et un seul
(AGAAGAAG) pour IL1403. La fixation de la protéine 3γ<sub>Ll</sub> sur ces motifs a été réalisée sur un ADN de
taille équivalente à ceux utilisés précédemment, et portant une succession du trinucléotide « GAA »
(Fig. 16B) permettant de retrouver les motifs AGAAGAAG et AAGAAGAA. La protéine 3γ<sub>Ll</sub> se fixe
spécifiquement et avec une forte affinité sur cette séquence puisque tout l'ADN est retardé. Un seul
complexe est observé et il apparaît stable puisqu'il n'est pas observé de profil caractéristique de
<figure><img src="image_122.png" /><figcaption>**Fig. 16.** A) Analyse bioinformatique du biais des octamères sur le chromosome des 2 sous-espèces de *L. lactis*. B) Interaction des protéines 3γ avec un fragment d'ADN contenant les 6 répétitions de triplet GAA.</figcaption></figure>
<figure><img src="image_123.png" /><figcaption>**Fig. 17.** Interaction des protéines 3γ avec un fragment d'ADN contenant un octamère candidat en monocopie. A) AGAAGAAG, B) AAGAAGAA, C) GGGCAGGG (motif canonique KOPS d'E. coli), D) localisation des motifs sur une région de 10 kb centrée sur *dif*<sub>SL</sub>, E) GAAGAAGA</figcaption></figure>

<figure><img src="image_124.png" /><figcaption>**Fig. 18.** A) Biais du motif GAAGAAG sur le chromosome de la souche IL1403. B) Interaction des protéines 3γ avec un fragment d'ADN contenant le motif GAAGAAG en monocopie.</figcaption></figure>
<figure><img src="image_125.png" /><figcaption>**Fig. 19.** Interaction des protéines 3γ avec un fragment d'ADN contenant trois KOPS (A) ou 3 motifs GAAGAAG (B). Localisation du motif GAAGAAG sur une région de 10 kb centrée sur *dif*<sub>SL</sub> du génome de la souche IL1403 (C).</figcaption></figure>
dissociation pendant l'électrophorèse. Le domaine y de la protéine FtsK de *L. lactis* reconnaît donc un motif contenu dans l'ADN (GAA)₆.

Afin de déterminer plus précisément le motif reconnu par la protéine 3γLI, nous avons réalisé des expériences de retard sur gel (EMSA) en présence d'ADN contenant un seul motif, soit AGAAGAAG (Fig. 17A), soit AAGAAGAA (Fig. 17B), soit GAAGAAGA (Fig. 17E). Des retards assez faibles, observés en absence de poly[dl-dC] comme pour l'interaction 3γEC / KOPS (Fig. 17C), sont visibles uniquement avec les motifs AGAAGAAG et GAAGAAGA. Sachant qu'aucun de ces motifs n'est présent dans la région de 10 kb centrée sur *dif*SL (Fig. 17D) et que le mot commun entre ces motifs est GAAGAAG, nous avons réalisé une analyse bioinformatique et des expériences de gel retard avec ce mot de 7 lettres. Ce motif est à la fois le plus surreprésenté et le plus biaisé des motifs de 7 lettres (Fig. 18A). De plus, le profil de fixation de la protéine 3γLI sur le motif GAAGAAG (Fig. 18B) est identique à celui observé avec les mots de 8 lettres. L'affinité des protéines 3γ pour un ADN contenant un seul motif étant faible, nous avons réalisé des tests de fixation en utilisant un ADN contenant trois motifs séparés par 6 pb (Fig. 19). L'ADN contenant 3 KOPS est entièrement retardé par la protéine 3γEC en absence et en présence d'ADN compétiteur, démontrant la forte affinité de la protéine pour cet ADN (Fig. 19A). Cependant, ce complexe est en grande partie dissocié par la présence de poly[dl-dC]. En revanche, la protéine 3γLI retarde entièrement cet ADN et forme des complexes stables, même en présence de poly[dl-dC] pour la concentration en protéine la plus grande (Fig. 19B). Ces résultats confirment donc que le sous-domaine y de la protéine FtsK de *L. lactis* reconnaît spécifiquement le motif GAAGAAG et qu'il y a très peu de fixation croisée entre 3γLI et les KOPS, ou entre 3γEC et GAAGAAG. La distribution de ce motif dans la région de 10 Kb centrée sur *dif*SL, révèle qu'il est présent cinq fois (Fig. 19C), ce qui est comparable à la représentation du motif KOPS chez *E. coli*. Toutefois, on peut noter qu'une copie est présente en orientation inverse de celle attendue pour diriger FtsKSL vers le site *dif*SL. Cependant, in vivo, l'effet d'un seul motif pourrait être moindre et ne pas gêner la translocation de FtsK jusqu'au site de recombinaison.

## 1.5) Conclusions-perspectives.

Nous avons mis en évidence chez *L. lactis* un motif interagissant fortement avec le sous-domaine y de la protéine FtsKSL, ce qui, par analogie avec les données récentes obtenues chez *E. coli* (Graham *et al.*, 2010), suggère fortement qu'il est impliqué dans la directionnalité de la translocation de la protéine. Ce motif de 7 pb, renommé SPOK pour *Streptococcal Polar sequence Orienting ftsK*, ne correspond pas à la séquence consensus GNGNAGGG des octamères de type KOPS/SRS/FRS, mais appartient à l'octamère le plus surreprésenté (5'-AGAAGAAG-3') dans la majorité des phyla bactériens (Davenport & Tummler, 2010). La taille du SPOK pose cependant la question du mode de fixation du sous-domaine y à ce motif, puisqu'un motif de 7 lettres est incompatible avec le modèle
de fixation déduit des données de co-cristallisation du sous-domaine γ de *P. aeruginosa* sur le KOPS canonique (5'-GGGCAGGG-3') (Löwe *et al.*, 2008). Ces données suggèrent en effet une fixation de trois sous-domaines γ à un site KOPS, les sous-unités A et C reconnaissant les deux triplets GGG distaux et la sous-unité B le dinucléotide central CA. Nous pouvons cependant exclure que le site « natif » corresponde soit à l'octamère 5'-AGAAGAAG-3' (octamère le plus biaisé sur les génomes des souches IL1403 et MG1363), voire au nonamère 5'-AAGAAGAAG-3', chaque domaine γLI se fixant au dinucléotide AG (ou au triplet AAG) (Fig. 20), car nous n'avons pas observé de différence d'interaction significative par gels retards (données non montrées).

<figure><img src="image_126.png" /><figcaption>**Fig. 20.** Biais d'orientation des motifs SPOK « étendus » sur le génome de la souche IL1403. Haut, GAAGAAG; centre, AGAAGAAG; bas, AAGAAGAAG. Le trait rouge indique la position du site *dif*<sub>SL</sub>. (réalisé à l'IBCG avec le programme oligoskew de S. Salzberg).</figcaption></figure>

Ainsi, *L. lactis* possède un système de translocation orientée du chromosome similaire aux γ-protéobactéries ou à *B. subtilis*, mais la nature des partenaires de la machinerie moléculaire est différente. Par analogie avec l'existence d'un système Xer atypique chez toutes les espèces de streptocoques, on peut se demander si les SPOK sont conservés chez ces espèces. Il est actuellement difficile de conclure car les données in silico semblent contradictoires. En effet, bien que l'orientation des motifs de type KOPS/SRS/FRS ne soit pas biaisée chez les autres espèces de streptocoques (données non montrées), alors que celle des SPOK l'est (données non montrées), les bactéries du
genre *Streptococcus* possèdent un sous-domaine FtsKγ très conservé et différent de celui des lactocoques (Fig. 21). De façon surprenante, ces sous-domaines possèdent les trois acides aminés impliqués dans la spécificité de reconnaissance des KOPS chez les γ-protéobactéries (Sivanathan *et al.*, 2009) (flèches rouges, Fig. 21)

<figure><img src="image_127.png" /><figcaption>**Fig. 21.** Alignement multiple des sous-domaines y des protéines FtsK/SpolIIIE de différentes espèces bactériennes. Les acides aminés importants pour la spécificité de reconnaissance des KOPS/SRS sont indiqués par des flèches rouges. L'analyse phylogénétique (programme NJ, bootstrap 1000) montre bien la proximité entre les sous-domaines y des streptocoques et des lactocoques.</figcaption></figure>
## Projet de recherche

Différents axes de recherches sont envisagés :

### 1) Rôle du système XerS/dif<sub>SL</sub> dans la résolution des dimères de chromosomes chez S. pneumoniae.

Si un faisceau d'évidences suggère que le système XerS/dif<sub>SL</sub> est impliqué dans la résolution des dimères de chromosomes chez *L. lactis* et *S. pneumoniae*, ceci n'a pas encore été démontré expérimentalement. Nous poursuivrons donc la caractérisation de XerS/dif<sub>SL</sub> en nous focalisant sur l'espèce *S. pneumoniae*. Ce choix est principalement liée au fait que de nombreux outils génétiques sont disponibles pour cette espèce et que c'est un des organismes modèles étudié de longue date au LMGM (équipe JP Claverts et P. Polard). Dans un premier temps, nous chercherons à démontrer l'existence d'une organisation monocistronique de XerS/dif<sub>SL</sub> en déterminant le point +1 de transcription de *xerS*. Des données préliminaires obtenues chez *L. lactis* (S. Nouaille-LISBP Toulouse, communication personnelle) indiquent que le site *dif* correspond à la boite -35 du promoteur *xerS* (*p*<sub>xerS</sub>). Ensuite, nous chercherons à démontrer l'implication de XerS/dif<sub>SL</sub> dans la résolution des dimères de chromosome. En utilisant la propriété particulière de *S. pneumoniae* à incorporer de l'ADN simple brin lors de sa transformation, nous générerons des dimères de chromosome par recombinaison interchromatide en utilisant comme substrat un fragment PCR correspondant à une borne d'inversion chromosomique et porteur d'un marqueur de sélection. Enfin, nous étudierons les conséquences phénotypiques de l'inactivation du système XerS/dif<sub>SL</sub>, en caractérisant les altérations morphologiques de différents mutants (*xerS*, dif<sub>SL</sub> ou *xers*-dif<sub>SL</sub>) générés par insertion d'un transposon mariner (Prudhomme *et al.*, 2006) par microscopie (photonique et électronique, collaboration S. Balor / plateau technique de microscopie de l'IBCG). Nous regarderons également les modifications de leur cinétique de croissance dans différentes conditions physiologiques (type de milieu, induction de la compétence) et dans différents contextes génétiques (doubles mutants *xerS/recA* et *xerS/comC0*, respectivement déficients pour la recombinaison ou pour la compétence). Ces analyses permettront d'identifier les processus biologiques perturbés par l'inactivation de la résolution des dimères de chromosomes.

### 2) Origine phylogénétique du module XerS/dif<sub>SL</sub>.

Une des questions posée par la structure particulière du module de recombinaison XerS/dif<sub>SL</sub>, similaire aux modules de recombinaison de certains prophages ou de certains ICEs, concerne l'origine évolutive de ce système. D'après « l'hypothèse de la complexité », qui postule que la transférabilité
d'un gène est inversement proportionnelle au nombre d'interactions de la protéine codée par le gène xénologue avec les protéines de la cellule hôte (Jain et al., 1999), l'absence d'interaction protéine-protéine directe entre FtsK et XerS pourrait constituer un argument supplémentaire pour considérer que le système XerS/dif<sub>SL</sub> ne correspond pas au système Xer ancestral, mais qu'il a été acquis par transfert horizontal chez l'ancêtre des *Streptococcaceae*. Convaincus que la famille des recombinases à tyrosines XerS n'était présente que chez les streptocoques / lactocoques, nous pensions qu'il était impossible de réaliser une analyse phylogénétique, vue la grande diversité de séquence des recombinases à tyrosine. L'analyse récente des bases de données de génomes bactériens semble indiquer que des recombinases proches de XerS existent dans d'autres familles de Firmicutes, notamment les genres *Lactobacillus* et *Bacillus* (B. Lafay, communication personnelle). Chez *Bacillus*, ces recombinases semblent préférentiellement associées à des plasmides (données non montrées). Nous réaliserons donc une analyse phylogénétique plus exhaustive des recombinases à tyrosine chez ces groupes bactériens, en regardant notamment la présence de sites dif-like à proximité des recombinases, afin d'identifier de nouveaux modules de recombinaison. Une étude fonctionnelle pourra alors être envisagée pour les candidats pertinents, notamment en utilisant la plateforme de recombinaison développée chez *E. coli* (Nolivos et al., 2010).

### 3) Caractérisation du couplage entre ségrégation des chromosomes et division cellulaire chez les streptocoques.

Chez *E. coli* et *B. subtilis*, l'inactivation du système Xer conduit à une filamentation des cellules, un phénotype propre au blocage du processus de division cellulaire, ainsi qu'un défaut de ségrégation des chromosomes (Fig. 22) (Sciochetti et al., 1999 ; Sciochetti et al., 2001).

<figure><img src="image_128.png" /><figcaption>**Fig. 22.** Morphologie cellulaire lors de l'inactivation des systèmes Xer. **a)** mutant *xerC* d'E. coli. **b)** différents mutants de *B. subtilis*. Les cercles rouges indiquent les défauts de ségrégation des chromosomes. **c)** mutant *xerS* de la souche *L. lactis* subsp. *cremoris* MG1363 en coloration LIVE/DEAD®. Les cellules mortes sont colorées en rouge. (Couverture de PLoS Genetics, juillet 2007).</figcaption></figure>
Bien que *L. lactis* soit capable de filamenter lors d'un blocage du processus de division cellulaire (Perez-Nunez *et al.*, 2011), l'inactivation du système XerS/difSL chez cette espèce provoque la formation de longues chaines (Fig. 22c), un phénotype plutôt caractéristique de l'inactivation des hydrolases du peptidoglycane impliquées dans la séparation des cellules (Buist *et al.*, 1995 ; Garcia *et al.*, 1999 ; Layec *et al.*, 2008 ; Layec *et al.*, 2009). Cette observation suggère fortement que les mécanismes responsables de la coordination entre résolution des dimères de chromosomes et division cellulaire sont différents chez les lactocoques. Comme un phénotype similaire a été observé pour les mutants xerS, difSL et ftsK chez *S. pneumoniae* (données non montrées), nous pensons que ces mécanismes de coordination seront conservés chez tous les streptocoques. Nous chercherons à caractériser le lien entre ségrégation chromosomique et division cellulaire chez *S. pneumoniae*, notamment le rôle de la protéine FtsK dans cette coordination. Le choix de *S. pneumoniae* est essentiellement motivé par le fait que c'est la seule espèce de streptocoques pour laquelle des données consistantes sur les mécanismes de division cellulaire existent (Morlot *et al.*, 2003 ; Noirclerc-Savoye *et al.*, 2003 ; Fadda *et al.*, 2003 ; Morlot *et al.*, 2004 ; Lara *et al.*, 2005 ; Zapun *et al.*, 2008 ; Maggi *et al.*, 2008). Dans un premier temps, nous étudierons les conséquences phénotypiques de l'inactivation du gène ftsK en caractérisant les altérations morphologiques de deux mutants exprimant respectivement une protéine tronquée de son domaine C-ter (mutant ftsKc), ou dépourvu d'activité FtsK (mutant ftsKNULL). Comme pour les mutants xerS, les altérations morphologiques des mutants ftsK seront analysées par microscopie photonique et électronique, et les modifications de leur cinétique de croissance dans différentes conditions physiologiques seront étudiées.

Une particularité intéressante de *S. pneumoniae* concerne l'obtention d'un mutant *ftsKNULL* viable. En effet, à moins d'utiliser des mutations compensatrices dans le gène *ftsA* (Geissler & Margolin, 2005) ou de surexprimer le gène *ftsN* (Draper *et al.*, 1998 ; Goehring *et al.*, 2007), il n'est pas possible d'obtenir un tel mutant chez *E. coli* car le domaine N-ter de la protéine est nécessaire à la formation du divisome (Draper *et al.*, 1998 ; Wang & Lutkenhaus, 1998), très probablement par sa capacité à interagir fortement avec FtsZ (Dubarry *et al.*, 2010). Ce résultat suggère un contrôle différent de la formation du divisome chez les streptocoques. Cette hypothèse semble confirmée par les différences observées dans le réseau d'interaction des protéines du divisome entre *E. coli* et *S. pneumoniae* (Maggi *et al.*, 2008), notamment l'absence d'interaction chez *S. pneumoniae* entre FtsK et FtsI (codant la peptidoglycane synthase septale PBP2x) (Fig. 23). Dans le but de mieux caractériser les étapes de formation du septum de division des streptocoques, et en complément des études morphologiques précédemment citées, nous caractériserons la localisation par microscopie à épifluorescence de différentes protéines du divisome (FtsZ, FtsK, FtsI), ou associées (LytB), fusionnées à la GFP (collaboration N. Campo, LMGM). L'utilisation de différents variants de la GFP
(YFP, CFP) permettra de réaliser des co-localisations de ces différentes protéines. Ces expériences seront réalisées dans différents contextes génétiques (souche sauvage, mutants *ftsKc* et *ftsKNULL*).

<figure><img src="image_129.png" /><figcaption>**Fig. 23.** Species-specific interaction profile among the nine cell division proteins common to *S. pneumoniae* and *E. coli*. (a) Interaction web among nine selected *S. pneumoniae* division proteins, FtsZ, FtsA, ZapA, FtsK, FtsQ/DivIB, FtsB/DivIC, FtsL, FtsW and FtsI/PBP2x, described in Table 2. (b) Interaction web among the nine *E. coli* cell division proteins, FtsZ, FtsA, FtsK, FtsQ/DivIB, FtsB/DivIC, FtsL, FtsN, FtsW and FtsI/PBP3 as described by Di Lallo *et al*. (2003), supplemented with the results reported in Table 3. Homodimerization is represented by circular arrows. The unbroken arrows indicate conserved interactions, while the dashed arrows indicate species-specific interactions. The double arrows indicate that heterodimerization was studied bi-directionally, with both possible constructions of fusion proteins: 434-X/P22-Y and 434-Y/P22-X.</figcaption></figure>

(d'après Maggi S. *et al*, 2008)
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Curriculum vitae

ECOLE DOCTORALE DE RATTACHEMENT : Biologie - Santé - Biotechnologies

Nom : Le Bourgeois

Prénom : Pascal

Né(e) le : 03 août 1964

Nationalité : Française

Fonctions : MCU classe normale, 9e échelon

I - PARCOURS UNIVERSITAIRE

1982 : Baccalauréat série D. PARIS.

1985 : DEUG B "Sciences de la nature et de la vie". Université François Rabelais - TOURS.

1986 : Licence de Biochimie. Université Paul Sabatier - TOULOUSE.

Licence de Biologie Cellulaire et Physiologie. Université Paul Sabatier - TOULOUSE.

1987 : Maîtrise de Biologie Cellulaire (mention Microbiologie). Université Paul Sabatier - TOULOUSE.

1989 : DEA de Microbiologie. Université Paul Sabatier - TOULOUSE.

1993 : Doctorat de l'Université Paul Sabatier, spécialité Microbiologie. Intitulé : "Structure et organisation du chromosome de Lactococcus lactis". Soutenue le 28 Mai 1993.

II - PARCOURS PROFESSIONNEL (après le doctorat)

1993 - 1994 : Attaché temporaire d'enseignement et de recherche, Université Paul Sabatier, TOULOUSE

1994 – prés.: Maître de conférences, Université Paul Sabatier, Toulouse. Titulaire du poste depuis septembre 1995.

III - ACTIVITES D'ENSEIGNEMENT (nature et niveaux des enseignements)

A l'université Paul Sabatier

1) Enseignement de biologie moléculaire (1994-2002).

Conjointement avec C. Cazaux (Pr. UPS), j'ai mis en place l'enseignement TD/TP de Biologie Moléculaire Appliquée. Cet enseignement, dispensé aux étudiants de L3 sur 4 journées entières (32h), avait pour objet de familiariser l'étudiant aux techniques classiques de biologie moléculaire.
L'objectif était de construire un plasmide navette procaryote-eucaryote portant un gène codant une triple fusion protéique (β-galactosidase/protéine Sh/thymidilate kinase) conférant un phénotype de résistance à l'ampicilline et à la zéomycine, et de sensibilité à l'AZT.

## 2) Enseignements de microbiologie industrielle (1994-2007).

J'ai mis en place l'enseignement de TD/TP du module de microbiologie industrielle pour l'IUP en utilisant comme base les TP créés par M. Lautier (MCF-UPS). Cet enseignement, dispensé aux étudiants L3 sur 4 journées entières (32h), se propose d'aborder les différentes problématiques liées à l'utilisation des bactéries lactiques en milieu industriel.

## 3) Enseignement de diagnostic moléculaire (1994-présent).

J'ai mis en place l'enseignement TD/TP de diagnostic moléculaire dispensé aux étudiants de M1. Cet enseignement se déroule sur 40h (une semaine continue de TD/TP) et a pour objet de sensibiliser les étudiants aux nouvelles méthodes de détection des contaminations microbiologiques. Il s'articule comme suit :

- utilisation de méthodes moléculaires d'identification de microorganismes (électrophorèse en champs pulsés, ARDRA, hybridation ADN-ADN)

- détection et quantification de microorganismes contaminants dans un produit alimentaire par PCR point final et PCR quantitative en temps.

## 4) Enseignements du M2P Diagnostic Microbiologique : approches innovantes (2007-présent)

Je participe à l'élaboration du programme de formation. J'enseigne en cours magistral (12h) sur les thèmes suivants : Taxonomie des organismes procaryotes, structure des génomes bactériens, méthodes de typage moléculaire, PCR quantitative en temps réel, nouvelles méthodes de séquençage. J'enseigne également en TD (dessin d'amorces PCR spécifiques d'espèces) et TP (optimisation de la qPCR pour la quantification de contaminants alimentaires).

## 5) Ateliers thématiques du M2R Microbiologie

Je participe régulièrement à un des ateliers du M2R de Microbiologie. Ceci consiste en un cours de 2h puis une évaluation de rapports écrits (minirevue ou projets de recherche) ou de soutenances.

**2009 :** Organisation, dynamique et plasticité des génomes microbiens et viraux.

**2008 :** Génomique comparative et nouvelles méthodes de séquençage.

**2007 :** Structure et plasticité des génomes bactériens.
**2005 :** Structure, plasticité et contraintes des génomes bactériens.

**2003 :** Structure des génomes bactériens et polarisation des éléments génétiques mobiles.

## Autres enseignements

**2003-2010 :** Interventions de 5h à l'INSA de Toulouse. "Génétique et taxonomie des bactéries lactiques". (Formation continu - CRITT).

**2005-2010 :** Intervention de 2h à l'école Doctorale Biologie-Santé-Biotechnologies de Toulouse (étudiants M2R, entrants INSERM) « La Radioprotection : Évaluation du risque & moyens de prévention ».

**2003 :** Intervention à l'école primaire de Saint Léon (Hte-Garonne). "Découverte des microorganismes responsables de la fermentation du raisin, manipulation des microscopes optiques".

**2001 :** Intervention de 3h à l'INSA de Toulouse. "Transferts génétiques chez les bactéries". (Formation continu - CRITT).

**1993-1997 :** Interventions à l'INRA de Rennes. "Principes de l'électrophorèse en champs pulsés : application à l'analyse des génomes procaryotes". (Formation continu des chercheurs INRA).

**Janv. 92 :** Intervention à l'ENSBANA de Dijon : "Méthodes de taxonomie moléculaire des procaryotes". (5ᵉ année d'école d'ingénieur).

# IV - ACTIVITES DE RECHERCHE

**13 publications dans des revues à comité de lecture, 5 chapitres d'ouvrages.**

## Publications dans des périodiques à comité de lecture

Passerini D, Beltramo C, Coddeville M, Quentin Y, Ritzenthaler P, Daveran-Mingot ML, and P Le Bourgeois (2010). Genes but not Genomes Reveal Bacterial Domestication of *Lactococcus lactis*. PLoS ONE 5(12): e15306.

Nolivos S, Pages C, Rousseau P, **Le Bourgeois P**, and F Cornet (2010). Are two better than one? Analysis of an FtsK/Xer recombination system that uses a single recombinase. Nucleic Acids Res. **38**:6477-6489.

**Le Bourgeois P**, Bugarel M, Campo N, Daveran-Mingot ML, Labonte J, Lanfranchi D, Lautier T, Pagès C, and P Ritzenthaler (2007). The Unconventional Xer Recombination Machinery of *Streptococci / Lactococci*. PLoS Genet. **3**(7): e117.
Campo N, Dias MJ, Daveran-Mingot ML, Ritzenthaler P, and **P Le Bourgeois** (2004). Chromosomal constraints in Gram-positive bacteria revealed by artificial inversions. Mol. Microbiol. **51**:511-522.

Campo N, Dias MJ, Daveran-Mingot ML, Ritzenthaler P, and **P Le Bourgeois** (2002). Genome plasticity in *Lactococcus lactis*. Antonie Van Leeuwenhoek. **82**:123-132.

Campo N, Daveran-Mingot ML, Leenhouts K, Ritzenthaler P, and **P Le Bourgeois** (2002). Cre-*loxP* recombination system for large genome rearrangements in *Lactococcus lactis*. Applied Environ. Microbiol. **68**:2359-2367.

**Le Bourgeois P**, Daveran-Mingot ML, and P Ritzenthaler (2000). Genome plasticity among related *Lactococcus* strains: Identification of genetic events associated with macrorestriction polymorphisms. J. Bacteriol. **182**: 2481-2491.

Daveran-Mingot ML, Campo N, Ritzenthaler P, and **P Le Bourgeois** (1998). A Natural Large Chromosomal Inversion in *Lactococcus Lactis* is Mediated by Homologous Recombination between Two Insertion Sequences. J. Bacteriol. **180**:4834-4842

**Le Bourgeois P**, Lautier M, van den Berghe L, Gasson MJ, and P Ritzenthaler (1995). Physical map of the *Lactococcus lactis* subsp. cremoris MG1363 chromosome: comparison with that of *Lactococcus lactis* subsp. lactis IL1403 reveals a large genome inversion. J. Bacteriol. **177**:2840-2850.

**Le Bourgeois P**, Lautier M, and P Ritzenthaler (1993). Chromosome mapping in lactic acid bacteria. FEMS Microbiol. Rev. **12**:109-124.

**Le Bourgeois P**, Lautier M, Mata M, and P Ritzenthaler (1992). Physical and genetic map of the chromosome of *Lactococcus lactis* subsp. lactis IL1403. J. Bacteriol. **174**:6752-6762.

**Le Bourgeois P**, Lautier M, Mata M, and P Ritzenthaler (1992). New tools for the physical and genetic map of *Lactococcus* strains. Gene. **111**:109-114.

**Le Bourgeois P**, Mata M, and P Ritzenthaler (1989). Genome comparison of *lactococcus* strains by pulsed-field gel electrophoresis. FEMS Microbiol. Letters. **59**:65-70.

Chapitres d'ouvrages

**Le Bourgeois P**, Langella P, and P Ritzenthaler (2000). Electrotransformation of *Lactococcus lactis*. In N. Eynard, and J. Teissié (eds.), Electrotransformation of Bacteria. pp. 65-70. Springer Lab Manual, Springer-Verlag, Heidelberg, Germany
Le Bourgeois P, Daveran-Mingot ML, and P Ritzenthaler (1998). Physical maps of *L. lactis* subsp. *lactis* IL1403 and *L. lactis* subsp. *cremoris* MG1363 chromosomes. *In* F.J. de Bruijn, J.R. Lupski, and G.M. Weinstock (eds.), Bacterial Genomes: Physical Structure and Analysis. pp. 669-672, Chapman & Hall, London.

Le Bourgeois P, Lautier M, Van den Berghe L, Serin G, Nedjari H and P Ritzenthaler (1995). Combined physical and genetic maps of *Lactococcus lactis* subsp. *lactis* IL1403 and *Lactococcus lactis* subsp. *cremoris* MG1363 chromosomes. Dev. Biol. Stand. **85**:597-603

Le Bourgeois P, Lautier M, Mata M, and P Ritzenthaler (1993). Cartographie génomique de quelques souches de laboratoire de *Lactococcus lactis*. Le lait. **73**:163-174.

Le Bourgeois P, Mata M, and P Ritzenthaler (1991). Pulsed-field gel electrophoresis as a tool for studying the phylogeny and genetic history of lactococcal strains. *in* G. M. Dunny, P. P. Cleary, and L. L. McKay (ed.), Genetic and Molecular Biology of Streptococci, Lactococci, and Enterococci, American Society for Microbiology, Washington, D.C. pp:140-145.

## 13 conférences (séminaires invités, communications orales). Présentations d'affiches non répertoriée (>30).

« Variabilité génétique et génomique chez *Lactococcus lactis* ». Ecole Thématique de Microbiologie Moléculaire, Presqu'île de Giens (France), 11-15 octobre 2010. **(sur comité de selection)**

“Unconventional Xer recombination in *Streptococci* and *Lactococci*”. 14th Conference on functional Genomics of Gram-Positive Microorganisms, Pise (Italie), 24-28 juin 2007. **(sur comité de selection)**

« Caractérisation d'un système alternatif pour la résolution des dimères de chromosomes chez les streptocoques ». 7ème Congrès National de la Société Française de Microbiologie. Nantes (France) 30, 31 mai et 1er juin 2007. **(Sur invitation)**

“Résolution des dimères de chromosome chez les Streptocoques”. UBLO, INRA, Jouy-en-Josas (France), 27 avril 2007. **(Sur invitation)**

“Exotic Xer recombination in *Streptococci* and *Lactococci*”. 7th ASM Conference on Streptococcal Genetics. St Malo (France), 18-21 juin 2006. **(sur comité de selection)**

“Inversions chromosomiques expérimentales chez les Firmicutes” VIe Congrès de la SFM. Bordeaux (France), 10-12 mai 2004. **(Sur invitation)**
"In silico and in vivo analysis of genome plasticity: applications to a non-model bacteria, *Lactococcus lactis*." Séminaire Algorithmique et Biologie, Quinzième série : Réarrangements Génomiques. Lyon (France), 2-4 2004. (Sur invitation)

"Evolution et contraintes de l'organisation du génome de *Lactococcus. Lactis*. URLGA, INRA, Jouy en Josas (France), 24 octobre 2002. (Sur invitation)

"Genome plasticity in *Lactococcus lactis*. Seventh Symposium on Lactic Acid Bacteria. Egmond aan Zee (Pays-Bas), 1-5 septembre 2002. (Sur invitation)

"Organisation et plasticité du génome de *Lactococcus lactis*. 8ᵉ Colloque du C.B.L. Dijon (France), 28-30 mai 1997. (sur comité de selection)

"Genome organization of some laboratory *Lactococcus* strains". Fourth Symposium on Lactic Acid Bacteria. Noordwijkerhout (Pays-Bas), 5-9 septembre 1993. (Sur invitation)

"Genome analysis of *Lactococcus lactis* by PFGE". AFRC, Institut of Food Research, Norwich (Angleterre). 22 mars 1991. (Sur invitation)

"Comparaison du génome de bactéries lactiques par électrophorèse en champs pulsés". Réunion du C.B.L. INRA de Jouy-en-josas (France). 8-9 septembre 1990. (Sur invitation)

V - ACTIVITES D'ENCADREMENT : liste nominative des personnes encadrées ou co-encadrées (rem. seuls ont été comptabilisés les étudiants de M2 et les doctorants [indiqués en gras])

**2008 :** Delphine Passerini (M2R Microbiologie – Toulouse, 8 mois). «Diversité génomique de *Lactococcus lactis* ».

**2006 - 08:** Delphine Passerini (Ingénieur d'étude sur contrat ANR, 2 ans). «Diversité génomique de *Lactococcus lactis* ».

**2006 - 10:** Sophie Nolivos (Doctorat de Microbiologie – Toulouse) « Mécanisme moléculaire de la recombinaison Xer chez les streptocoques ». Thèse soutenue le 17/09/2010. Co-direction à 50% avec François Cornet (DR CNRS, LMGM).

**2005 :** Marie Bugarel (M2R Microbiologie – Toulouse, 8 mois). « Caractérisation de mutants *xer* chez *Streptococcus pneumoniae* ».

**2003:** Emmanuelle Sacco (DEA Microbiologie – Toulouse, 8 mois). « Analyse transcriptionnelle des gènes impliqués dans la résolution des dimères de chromosomes chez *Lactococcus lactis* ».
2001-06: Miguel Dias (Doctorat de Microbiologie - Toulouse) « Plasticité du génome de *L. lactis*: domaines de contraintes révélés par inversions expérimentales ». Thèse soutenue le 22/03/2006. Co-direction à 50% avec Paul Ritzenthaler (Pr. UPS, LMGM).

2001: Miguel Dias (DEA Microbiologie – Toulouse, 8 mois). « Inversions chromosomiques chez *L. lactis* ».

2000: Alhousseynou Sall (DEA de microbiologie – Toulouse, 8 mois).

1997: Olivier Bois (DEA de Microbiologie – Toulouse, 8 mois) « Plasticité du génome des bactéries à Gram+: études des inversions chromosomiques chez *L. lactis* ».

1996-01: Nathalie Campo (Doctorat de Microbiologie - Toulouse) « Stabilité du génome des bactéries lactiques: étude d'inversions chromosomiques chez *Lactococcus lactis* ». Thèse soutenue le 26/02/2001. Co-direction à 50% avec Paul Ritzenthaler (Pr. UPS, LMGM).

1996: Nathalie Campo (DEA de Microbiologie – Toulouse, 8 mois) et Fabrice Sabathe (ESGBMA – Marseille, 8 mois). « Stabilité du génome des bactéries lactiques: étude d'inversions chromosomiques chez *Lactococcus lactis* ».

1993: Guillaume Serin (DEA de Microbiologie – Toulouse, 8 mois) et Hafed Nedjari (DEA Microbiologie – Caen, 8 mois) « Etude de l'organisation génomique de différentes souches de *Lactococcus lactis* ».

1993: Loïc van den Berghe (DEA de Microbiologie – Toulouse, 8 mois). "Construction d'une carte physique du chromosome de *Lactococcus lactis* subsp. cremoris MG1363".

1991: Jérôme Sieurin (BE 5e année INSA – Toulouse, 5 mois). "Localisation du site *OriC* du chromosome de *Lactococcus lactis* par synchronisation cellulaire".

1990: François-Yves Bouget. (DEA de Microbiologie – Toulouse, 5 mois). "Construction d'une banque d'ADN génomique de *Lactococcus lactis*".

VI - AUTRES

- Responsable pédagogique du « L3 Bioingénierie » depuis 1998.

- Coresponsable du M2P « Diagnostic Microbiologique: approches innovantes » depuis 2007 (date de sa création).

- Présidence de jury de Bac (tous les 3 ans).

- Membre de la commission de spécialiste (65e section) de 1998 à 2000.
- Personne Compétente en Radioprotection des UMR5100 et UMR5099 (IBCG-CNRS) de 1999 à 2010.

- Membre du comité scientifique et du comité d'organisation du congrès national « Colloque des Bactéries Lactiques » du 27 au 29 mai 2009.

- Reviewer d'articles scientifiques (Microbiology, Mol. Microbiol., BMC Genomics)

- Expert de l'appel d'offre pour l'attribution d'allocations doctorales et postdoctorales par le DIM "Maladies infectieuses, parasitaires et nosocomiales émergentes" du Conseil régional Ile-de-France (2008).

- Coresponsable d'un « Workpackage » du programme ANR « Génoferment » (ANR-05-PNRA-20)