<table><caption>TABLE 1. Bacterial strains and plasmids used in this study</caption><thead><tr><th>Strain or plasmid</th><th>Relevant property(s)<sup>a</sup></th><th>Source or reference</th></tr></thead><tbody><tr><td><strong>Strains</strong></td><td></td><td></td></tr><tr><td><em>L. lactis</em></td><td></td><td></td></tr><tr><td>MG1363</td><td>Plasmid-free strain</td><td>12</td></tr><tr><td>NZ9000</td><td>MG1363 <em>pepN::nisRK</em></td><td>21</td></tr><tr><td>NZ9700</td><td>Nisin-producing transconjugant containing Tn5276</td><td>20</td></tr><tr><td>CL307/1</td><td>NZ9000, <em>Em<sup>r</sup></em>, chromosome::(pCL307)<sub>n</sub></td><td>This work</td></tr><tr><td>CL307/1-311</td><td>CL307/1, <em>Spc<sup>r</sup></em>, <em>pcp</em> region::(pCL311)<sub>n</sub></td><td>This work</td></tr><tr><td>CL307/5</td><td>NZ9000, <em>Em<sup>r</sup></em>, chromosome::(pCL307)<sub>n</sub></td><td>This work</td></tr><tr><td>CL307/5-311</td><td>CL307/5, <em>Spc<sup>r</sup></em>, <em>pcp</em> region::(pCL311)<sub>n</sub></td><td>This work</td></tr><tr><td>CL307/1-324</td><td>CL307/1, <em>Spc<sup>r</sup></em>, <em>ilvD</em>::(pCL324)<sub>1</sub></td><td>This work</td></tr><tr><td><em>E. coli</em></td><td></td><td></td></tr><tr><td>DH5α</td><td>F<sup>-</sup> φ80<em>lacZΔM15 Δ(lacZYA-argF)U169 endA1 recA1 hsdR17(r<sub>K</sub><sup>-</sup> m<sub>K</sub><sup>+</sup>) deoR thi-1 supE44 λ<sup>-</sup> gyrA96 relA1</em></td><td>Life Technologies</td></tr><tr><td>EC1000</td><td>MC1000 <em>glgB::repA</em></td><td>28</td></tr><tr><td><strong>Plasmids</strong></td><td></td><td></td></tr><tr><td>pLS1</td><td><em>Tc<sup>r</sup></em>, broad-host-range plasmid</td><td>37</td></tr><tr><td>pILOX1204</td><td><em>Ap<sup>r</sup></em>, reporter plasmid for measuring Cre recombinase activity; corresponds to pIL253 with P59-<em>loxP1</em>-T<sub>1</sub>T<sub>2</sub>T<sub>rep</sub>-<em>loxP2-lacZ</em> cloned into the multiple cloning site</td><td>K. J. Leenhouts, laboratory collection</td></tr><tr><td>pRL1</td><td><em>Em<sup>r</sup></em>, 5-kb integrative vector</td><td>25</td></tr><tr><td>pR324</td><td><em>Ap<sup>r</sup></em>, <em>Em<sup>r</sup></em>, <em>Spc<sup>r</sup></em>, <em>aad9</em> in pR244</td><td>9</td></tr><tr><td>pCL50</td><td><em>Ap<sup>r</sup></em>, pBSIIKS(-) (Stratagene) with an I-CeuI site inserted at the <em>AflIII</em> site</td><td>Laboratory collection</td></tr><tr><td>pCL52</td><td><em>Em<sup>r</sup></em>, 2.4-kb cloning vector; corresponds to pCL50 where <em>bla</em> gene and phage f1 ori are replaced by the <em>ermAM</em> gene</td><td>This work</td></tr><tr><td>pCL57</td><td><em>Spc<sup>r</sup></em>, 2.45-kb cloning vector; corresponds to pCL50 where <em>bla</em> gene and phage f1 ori are replaced by the <em>aad9</em> gene</td><td>This work</td></tr><tr><td>pCL60</td><td><em>Em<sup>r</sup></em>, 3.26-kb transpositional vector; <em>ISS1</em> insertion sequence cloned at the <em>Sap1</em> site of pCL52</td><td>This work</td></tr><tr><td>pCL302</td><td><em>Spc<sup>r</sup></em>, pCL57 with <em>loxP1</em> cassette cloned at the <em>BamHI</em> site</td><td>This work</td></tr><tr><td>pCL307</td><td><em>Em<sup>r</sup></em>, pCL60 with <em>loxP2</em> cassette cloned at the <em>EcoRI</em> site</td><td>This work</td></tr><tr><td>pCL311</td><td><em>Spc<sup>r</sup></em>, pCL302 with a 4.5-kb fragment of <em>pcp</em> region cloned at the <em>EcoRI</em> and <em>HindIII</em> sites</td><td>This work</td></tr><tr><td>PCL324</td><td><em>Spc<sup>r</sup></em>, pCL302 with a 2.8-kb fragment of <em>ilv</em> operon cloned at the <em>HindIII</em> site</td><td>This work</td></tr><tr><td>pGh9</td><td><em>Em<sup>r</sup></em>, 3.7-kb cloning vector with a thermosensitive replicon [<em>repA</em> (Ts)]</td><td>31</td></tr><tr><td>pNZ8048</td><td><em>Cm<sup>r</sup></em>, 3.4-kb expression vector</td><td>11</td></tr><tr><td>pNG8048-Cre</td><td><em>Cm<sup>r</sup></em>, pNZ8048 with <em>cre</em> gene cloned at the <em>NcoI</em> and <em>HindIII</em> sites</td><td>K. J. Leenhouts, laboratory collection</td></tr><tr><td>pGh-Cre</td><td><em>Cm<sup>r</sup></em>, 4.8-kb pNG8048-Cre derivative with thermosensitive replicon of pGh9</td><td>This work</td></tr></tbody></table>

recombinase Cre and demonstrated the usefulness of this approach by generating three large inversions that modify the genome organization to different extents.

## MATERIALS AND METHODS

**Bacterial strains and growth conditions.** The strains used in this work are listed in Table 1. *L. lactis* strains were grown at 30°C in M17 (38) broth (Merck, Darmstadt, Germany) supplemented with 0.5% glucose (GM17). *E. coli* was grown at 37°C in Luria-Bertani broth (Difco Laboratories, Detroit, Mich.). Antibiotics were used at the following concentrations for *E. coli*: erythromycin (Em), 150 µg/ml; chloramphenicol (Cm), 10 µg/ml; spectinomycin (Spc), 100 µg/ml; tetracycline (Tc), 2 µg/ml; and kanamycin, 40 µg/ml. For *L. lactis*, the concentrations were as follows: Em, 5 µg/ml; Cm, 5 µg/ml; Spc, 200 µg/ml; and Tc, 2 µg/ml.

Growth kinetics were determined in GM17 broth as follows. Culture tubes (diameter, 16 mm) containing 5 ml of prewarmed medium were inoculated with 2% of an overnight culture and incubated at 30°C without shaking in a water bath. Bacterial growth was monitored by spectrophotometric measurements of the optical density at 600 nm (model UV-1205; Shimadzu, Kyoto, Japan) every 30 min until the culture reached the stationary phase. The growth curve of each strain was constructed from the results of six to eight independent experiments.

**DNA manipulations.** Restriction enzymes, Klenow polymerase, Vent polymerase, and T4 DNA ligase were purchased from either Roche Molecular Biochemicals (Mannheim, Germany) or New England Biolabs (Beverly, Mass.) and used

as recommended by the suppliers. *L. lactis* strains were electrotransformed according to the method of Le Bourgeois et al. (24) with 5 µg of integrative plasmid or 50 ng of the replicative plasmid pGh-Cre and plated onto GM17 plates containing the appropriate antibiotic (Em, Spc, or Cm). *E. coli* DH5α was electrotransformed by use of a gene pulser device (Bio-Rad, Richmond, Calif.) according to the manufacturer's recommendations. *E. coli* plasmid DNA was isolated by use of a Qiaprep spin kit (small scale) or a plasmid midi kit (large scale) (Qiagen GmbH, Hilden, Germany) according to the manufacturer's instructions. Preparation of lactococcal genomic DNA embedded in agarose matrix, the PFGE method, and Southern hybridizations on dried gels were performed as previously described (26). Hybridization signals were detected with a bioimaging analyzer BAS1000 system (Fuji Photo Film Co., Tokyo, Japan) and analyzed with Tina version 2.07c software (Raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany).

**Plasmids.** The plasmids used in this work are listed in Table 1. The *bla* gene and the phage f1 replication origin of pCL50 were replaced by different antibiotic resistance genes as follows (in all cloning steps, blunt-ended fragments were generated by using Klenow polymerase before ligation). The 1,464-bp *SspI/BspHI* fragment (containing the colE1 replication origin and the *lacZ* peptide α regions) of pCL50 was ligated to the 919-bp *DdeI/AccI* fragment of pAMβ1 (the wild-type *ermAM* gene) (32) to give pCL52 and to the 983-bp *NdeI/XbaI* fragment of pR324 (the wild-type *aad9* gene from pDL269) (22) to give pCL57. A 934-bp DNA fragment containing the functional *ISS1* insertion sequence was amplified by PCR from pRL1 with the following primers: *ISS1-5* (TGAAAAA TTTACAATTCTACTATCA) and *ISS1-3* (CGCTTATTTGGACGACAAT

<footnote>a Ap, ampicillin.</footnote>
GA) (Eurogentec, Seraing, Belgium). The fragment was then ligated with *Sap*I-digested pCL52 to give the transpositional integrative plasmid pCL60.

pGh-Cre was obtained by ligating a 2.3-kb *Acc*I fragment (corresponding to the *cat* and the *P*<sub>nisA</sub>-*cre* genes) from pNG8048-Cre to a 2.55-kb *Xba*I-*Hind*III fragment [corresponding to the *ori*(Ts) region] of pGh9. Transformation, selection, and propagation of pGh-Cre were performed in *E. coli* EC1000. pGh-Cre can replicate in *L. lactis* at 28°C but not at 37°C.

**Construction of loxP cassettes.** The 1,187-bp *loxP1* cassette (P59-*loxP*-*T*₁*T*₂*T<sub>rep</sub>) from pILOX1204 was amplified by PCR with the following primers: P59lox1T1T2-1 (CGGGATCCATGATGTTGTTTCTAAA) and P59lox1T1T2-2 (CTCAGGATCCATCGCAACATCAAA). The PCR product was then digested with *Bam*HI (underlined sequences) and cloned into the *Bam*HI site of the integration plasmid, pCL57, to generate pCL302. The *loxP2* cassette (*T*₁*T*₂*T<sub>rep</sub>-*loxP*-*tetL* open reading frame [ORF]) was constructed in a two-step cloning procedure: a 649-bp DNA fragment containing the three transcription terminators and a *loxP* site from pILOX1204 was first amplified by PCR with the primers T1T2lox2-1 (GGAATTCCCCCTGTTTTGGCGGATGAGA) and T1T2lox2-2 (CCTCCGGATCCTAGCGACAATAACTT). The PCR product was then digested with *Bam*HI (underlined sequence in T1T2lox2-2) and ligated to a 1,495-bp DNA fragment (corresponding to the *tetL* ORF with its own transcription terminator) that had been generated by PCR amplification of pLS1 with the primers Tetra3 (CCCTATAAACTACAGATCTGCCCTCATTAT) and Tetra2 (GCCCTCTTGAATTCCTGTT) and digested with *Bgl*II (underlined sequence in Tetra3). The resulting 2,144-bp fragment (*T*₁*T*₂*T<sub>rep</sub>-*loxP*-*tetL* ORF) was amplified by PCR from an aliquot of the ligation mixture with primers T1T2lox2-1 and Tetra2, digested with *Eco*RI (boldface sequences in T1T2lox2-1 and Tetra2), and cloned into the *Eco*RI site of pCL60 to generate pCL307.

**Chromosomal location and orientation of integrated plasmids.** All of the integrative plasmids described in this study contain unique *Apa*I, *Sma*I, *Not*I, and *I-Ceu*I restriction sites. Their integration sites, as well as their orientations, on the chromosome of lactococcal strains were determined by Southern hybridization of PFGE-separated chromosomal restriction fragments as previously described (25).

**Induction of the *cre* gene by nisin.** Strains carrying pGh-Cre were grown overnight at 28°C in GM17-Cm, diluted 100-fold in the same medium supplemented with a 1:1,000 dilution of the supernatant of the nisin-producing strain NZ9700 (20), and allowed to grow at 28°C to an optical density at 600 nm of 1.0.

**Stabilization of chromosomal rearrangements.** The loss of pGh-Cre was provoked as described by Maguin et al. (31) with minor modifications: strains containing pGh-Cre were grown overnight in GM17-Cm medium at 28°C, diluted 100-fold in 5 ml of GM17 without antibiotics, and incubated for 2 h at 28°C (water bath) to allow exponential growth to resume. The cultures were then shifted to 37.5°C for 7 h (water bath), and 50 µl of a 10⁻⁵ dilution was plated on GM17 plates (nonselective medium) and incubated at 28°C. One hundred colonies were transferred with toothpicks to nonselective or Cm-containing GM17 plates and incubated at 28°C. Approximately 25% of the colonies were phenotypically Cm*, and the loss of pGh-Cre plasmid was confirmed by Southern hybridization with the *cre* gene as a probe. The genomic structure (parental type or inverted) of the Cm* colonies was determined by PFGE and Southern hybridization by use of the *tetL* ORF probe.

## RESULTS

**Scheme for generation of chromosomal inversions by use of the inducible Cre-*loxP* recombination system.** The general scheme of the strategy (Fig. 1) involved a three-step procedure. The first step was the integration of two inverse-order *loxP* cassettes into the chromosome of *L. lactis* subsp. *cremoris* strain NZ9000, either randomly or at a fixed point depending on the integrative plasmid used. Strain NZ9000 is a derivative of strain MG1363, in which the *nisRK* constitutive operon was integrated into the dispensable gene *pepN*, allowing the use of the nisin induction-controlled expression system (21). Briefly, this food-grade gene expression system is based on the use of the promoter of the *nisA* gene (the structural gene of nisin), PnisA, which is inducible by the addition of nisin via the two-component regulatory system consisting of the response regulator protein NisR and the nisin sensor histidine kinase NisK. In nonproducing nisin strains that contain the *nisRK* operon,

<figure><img src="image_68.png" /></figure>

FIG. 1. Schematic representation of the Cre-*loxP* recombination system. A, B, C, and D represent the chromosome order. The letter n represents the number of AU of the integrated plasmids. The *loxP* sites, represented by triangles, are drawn in inverse orientation in order to yield an inversion after Cre-mediated recombination. The other option, whereby the *loxP* sites would be oriented as direct repeats, has been omitted from the figure.

genes under the control of PnisA are expressed at a very low level. When nisin is added to the growth medium, transcription of these genes is activated. The physical chromosome map of NZ9000 is thought to be identical to that of MG1363 (26), because no restriction pattern variations were revealed by PFGE after *Apa*I, *Sma*I, *Not*I, or *I-Ceu*I digestions (data not shown). One *loxP* cassette, the *loxP1* cassette (Fig. 2A), consisted of the lactococcal constitutive promoter P59 (40) followed by a *loxP* site and three lactococcal transcription terminators (*T*₁*T*₂*T<sub>rep</sub>). The second cassette, the *loxP2* cassette (Fig. 2A), corresponded to the three transcription terminators, *T*₁*T*₂*T<sub>rep</sub>, followed by a *loxP* site and the promoterless *tetL* ORF from pLS1 (37). The function of the terminators in the
<figure><img src="image_69.png" /></figure>

<figure><img src="image_70.png" /></figure>

<figure><img src="image_71.png" /></figure>

<figure><img src="image_72.png" /></figure>

<figure><img src="image_73.png" /><figcaption>FIG. 2. (A) Structures of the *loxP* cassettes used in the Cre-*loxP* recombination system. The *loxP* sites are represented by triangles. (B) Plasmids used for the Cre-*loxP* recombination. Abbreviations: N, NotI; C, I-CenI; A, ApaI; H, HindIII; E, EcoRI; S, SmaI; Ac, AccI; X, XbaI. (C) Schematic representation of the chromosomal organization of strain NZ9000 showing the location of the inversion endpoints. Different gray scales indicate inverted fragments. Arrows indicate the orientation of the *loxP* sites.</figcaption></figure>
*loxP1* cassette was to stop transcription from the P59 promoter, and the *loxP2* cassette terminators prevented any readthrough from a chromosomal promoter that could activate expression of the *tetL* ORF. Integration of the *loxP* cassettes into the chromosome via replicative transposition (ISSI transposition) or single crossing-over (homologous recombination) generally gives rise to multicopy insertion of the integrative plasmids (8, 27, 29, 31).

The second step was the transformation of the double integrant (containing the *loxP1* and *loxP2* sites in its chromosome) with the thermosensitive replication plasmid pGh-Cre (Fig. 2B), which contains the *cre* gene regulated by the inducible promoter PnisA (11). In permissive conditions for pGh-Cre replication and *cre* expression, the Cre recombinase catalyzes recombination between two *loxP* sites. The result of the recombination depends on the relative orientation of the *loxP* sites: if they are oriented as direct repeats, the recombination event leads to the deletion of the DNA segment between the sites, and if they are oriented as inverse repeats, the DNA segment between the sites is inverted.

The last step was the recovery of cells harboring either parental- or inverted-type chromosome structures. This was achieved by removing pGh-Cre by growing bacteria at 37.5°C, a temperature not permissive for pGh-Cre replication. Cells that acquired the inverted-type chromosome could be screened for their ability to grow in the presence of Tc, because the recombination between the *loxP1* and *loxP2* sites generated a transcriptional fusion between the P59 promoter and the *tetL* ORF. To illustrate the usefulness of this genetic system, we constructed large inversions at four positions in the NZ9000 chromosome (Fig. 2C).

**Construction of a 500-kb inversion in the same replication arm of the NZ9000 chromosome.** (i) **Integration of the *loxP*** cassettes. The transpositional plasmid pCL307 (Fig. 2B) was first integrated at random into the chromosome of NZ9000. The pCL307 integration sites of 40 Em<sup>r</sup> clones were located and oriented on the physical map of the NZ9000 genome. The chromosomal structure of one of them, named CL307/1 (Fig. 3A, panel 1), which was localized near the *oriC* site (Fig. 2C), was analyzed after I-CeuI digestion and hybridization with the *tetL* ORF as a probe. In addition to the 180-kb hybridizing fragment representative of the pCL307 integration, a 5.4-kb fragment was observed (Fig. 3B, lane 1). This hybridizing fragment corresponded to plasmid amplification at an undetermined copy number, but the amplification unit (AU) number was found to vary from 1 to 50 depending on the integration event (N. Campo, unpublished data). The integrative plasmid pCL311 (Fig. 2B) was then inserted into the chromosome of strain CL307/1, generating strain CL307/1-311. Plasmid pCL311 integrates at the *pcp* locus by homologous recombination, and its correct integration site, together with the resulting chromosomal structure (Fig. 3A, panel 2), was verified by PFGE after *Apa*I, *Sma*I, and I-CeuI digestion and hybridization with *pcp* or pBSIIKS as a probe (data not shown). An amplification of integrated pCL311 plasmid has been also observed (data not shown).

(ii) **Cre-mediated recombination between the *loxP*** sites. Strain CL307/1-311 was transformed by the pGh-Cre plasmid. The resulting strain was cultured in conditions permissive for plasmid replication and used to determine the optimal exper-

imental conditions for Cre-mediated recombination. First, the AU numbers of pCL307 and pCL311 were monitored during growth for 100 generations with or without antibiotics by Southern analysis of I-CeuI-digested chromosomes with a pBSIIKS probe. In the absence of pGh-Cre, the AU number was relatively stable, regardless of the growth conditions (Fig. 4, left lanes). As expected, the presence of pGh-Cre led to a strong decrease in the AU copy number, but only under non-selective growth conditions (Fig. 4, right lanes), suggesting that plasmid amplification was necessary to confer an Em and Spc resistance phenotype to the strain. Chromosomal inversion by recombination between the *loxP1* and *loxP2* sites generated a new 330-kb I-CeuI fragment (Fig. 3A, panel 4) that was identified by hybridization with *tetL* as a probe. This property was used to evaluate the recombination efficiency between *loxP1* and *loxP2* sites during growth without selective pressure. Unexpectedly, induction of the *cre* gene reduced the recombination efficiency between the *loxP* sites (data not shown). A hypothesis to explain this behavior is given in Discussion. Altogether, these results demonstrated that the most optimal experimental conditions for Cre-mediated recombination between the direct- or inverse-order *loxP* sites were the noninduction of the *cre* gene and nonselective growth of the cells (i.e., the absence of the antibiotics Em and Spc). Under these conditions, the ratio of the hybridization signals of the 330- and 180-kb I-CeuI fragments allowed us to estimate that 15% of the chromosomes of the cell population contained the 500-kb inversion (Fig. 3B, lane 2).

(iii) **Isolation of strains with a parental- or inverted-type chromosome.** Strains carrying either the parental (Fig. 3A, panel 3)- or the inverted (Fig. 3A, panel 4)-type chromosome were isolated after removal of pGh-Cre by growth at a non-permissive temperature for its replication. Although inverted-type strains can be selected for, their ability to grow in the presence of Tc, the high recombination efficiency allowed the recovery of parental- and inverted-type strains directly from colonies obtained from GM17 plates at 37°C without selective pressure after I-CeuI digestion of individual chromosomal DNA, PFGE separation, and hybridization with *tetL* as a probe (Fig. 3B, lanes 3 and 4). It should be pointed out that every parental- and inverted-type strain analyzed at this step contained only one copy of each *loxP* cassette (data not shown), indicating that the removal of AU by recombination between the proximal direct-order *loxP* sites has an efficiency of 100%.

**Inversions of half of the *L. lactis* chromosome in two different regions.** Two other large chromosomal inversions were constructed through the same experimental procedure. First, a 1,160-kb inversion was generated by integration of pCL311 in the *pcp* locus of the strain CL307/5 (Fig. 2C). This inversion of half of the chromosome altered neither the length of the two replication arms nor the location and orientation of genes with respect to *oriC*. The proper integration of these plasmids and the resulting chromosomal structure of the double integrant were verified by Southern hybridation with *tetL*, *pcp*, or ISSI probes (data not shown). Both plasmids were integrated into the chromosome in multiple copies. In addition to the removal of the amplification units of the pCL307 and pCL311 plasmids, the introduction of pGh-Cre into the double integrant led to chromosomal inversion in 50% of the cell population, as de-
A

1

<figure><img src="image_74.png" /></figure>

2

<figure><img src="image_75.png" /></figure>

3

<figure><img src="image_76.png" /></figure>

4

B

<figure><img src="image_77.png" /><figcaption>FIG. 3. (A) Theoretical chromosome structures of strain CL307/1 (panel 1), strain CL307/1-311 (panel 2), and parental- (panel 3) and inverted (panel 4)-type strains. The predicted sizes of the I-Ceu1 restriction fragments are indicated, and those hybridizing with the *tetL* probe are indicated in boldface. Fragment sizes are not drawn to scale. C, I-Ceu1. (B) Hybridization of I-Ceu1-digested chromosomes with the *tetL* probe after separation by PFGE (electrophoresis conditions were 10 V/cm and 17 s of pulse time for 13 h). Lanes: M, lambda DNA concatemer size standards; L, HindIII-digested lambda DNA; 1, CL307/1-311; 2, CL307/1-311 × pGh-Cre in permissive replication conditions; 3, parental-type strain; 4, inverted-type strain. Hybridization was performed in the presence of ³²P-labeled lambda DNA.</figcaption></figure>
<figure><img src="image_78.png" /><figcaption>FIG. 4. Visualization of the pCL307 and pCL311 amplification units in strains CL307/1-311 and CL307/1-311 × pGh-Cre in different conditions by hybridization of PFGE-separated chromosomal DNA after *ApaI* digestion with pBSIIKS(−) as a probe. The number above each lane indicates the generation number. The signs − and + correspond to the absence and presence, respectively, of Em and Spc in the growth medium.</figcaption></figure>

termed by the ratio of hybridization signals from fragments that signed the inverted or parental structure (Fig. 5, lane 1), and parental- or inverted-type strains were easily isolated after elimination of the pGh-Cre plasmid (Fig. 5, lanes 2 and 3).

Second, a 1,115-kb inversion, which largely modifies the length of the two replication arms by shifting the *terC* site at 180 kb to the *oriC* position (Fig. 2C), was constructed by transforming strain CL307/1 with pCL324 (Fig. 2B), a plasmid able to integrate at the *ilv* locus of the NZ9000 chromosome. pCL324 was integrated in a single copy into the CL307/1 chromosome (data not shown). Introduction of pGh-Cre into the double integrant led to a recombination event between the different *loxP* sites, removing the AU of pCL307 from every cell in the population and inverting the chromosomal DNA located between the inverse-order *loxP1* and *loxP2* sites in 6% of the population (data not shown). The proper chromosomal structures of parental- and inverted-type strains were confirmed by Southern analysis with the *tetL* ORF as a probe after I-CeuI digestion and separation by PFGE (data not shown).

**Effect of the chromosomal inversion on the growth rate in rich medium.** In *E. coli* and *Salmonella enterica* serovar Typhimurium, large chromosomal inversions can affect the fitness of the cells to various extents depending on the positions of their endpoints (15, 30, 35). Each inversion constructed in this study was evaluated for a direct effect on cell fitness by comparing the growth rates of the parental- and inverted-type strains in GM17. All inverted-type strains have a reduced growth rate compared to that of their isogenic parental-type counterpart (Table 2), with a decrease in the specific growth rate ranging from 8% for the strains with the 1,160-kb inversion that altered neither the length of the two replication arms nor the location and orientation of genes with respect to *oriC* to 18% for the strains with the 1,115-kb inversion that largely modified the lengths of the two replication arms.

DISCUSSION

With the aim of studying the dynamics of the *L. lactis* chromosome, we proposed to experimentally construct large inversions from different locations of the chromosome by using the homologous recombination strategy described for *Bacillus subtilis* (39). Unfortunately, we observed that single crossing-over and transpositional integration of exogenous DNA in *L. lactis*

<figure><img src="image_79.png" /><figcaption>FIG. 5. Hybridization of *ApaI*-digested chromosomes with the *tetL* probe after separation by PFGE (electrophoresis conditions were 10 V/cm and 7 s of pulse time for 13 h). Lanes: M, lambda DNA con-catemer size standards; L, HindIII-digested lambda DNA; 1, CL307/5-311 × pGh-Cre in permissive replication conditions; 2, parental-type strain isolated after pGh-Cre curing; 3, inverted-type strain isolated after pGh-Cre curing. Hybridization was performed in the presence of ³²P-labeled lambda DNA. The letter I corresponds to the *ApaI* fragment that signs the inverted structure, whereas P corresponds to the *ApaI* fragment characteristic of the parental structure.</figcaption></figure>

generally lead to the amplification of the integrated DNA, regardless of the selective marker used (data not shown). Similar observations have been made by different groups (8, 27, 29, 31). As inversions can only be easily obtained if the two targets are both present on the chromosome in single copies, this amplification phenomenon constituted a major drawback. To overcome this difficulty, we devised new genetic tools based on the use of the recombinase Cre from *E. coli* phage P1 (1) to obtain chromosomal inversions between two inverse-order *loxP* sites integrated at various positions on the chromosome of *L. lactis*. Paradoxically, the Cre-*loxP* recombination system is

<table><caption>TABLE 2. Specific growth rates of *L. lactis* strains in GM17 medium</caption><thead><tr><th rowspan="2">Inversion interval</th><th colspan="2">Specific growth rate (per hour) ± SE with indicated chromosomal structure</th></tr><tr><th>Parental</th><th>Inverted</th></tr></thead><tbody><tr><td>307/1-pcp</td><td>1.35 ± 0.03</td><td>1.15 ± 0.03</td></tr><tr><td>307/5-pcp</td><td>1.33 ± 0.04</td><td>1.23 ± 0.08</td></tr><tr><td>307/1-ilvD</td><td>1.30 ± 0.03</td><td>1.09 ± 0.04</td></tr></tbody></table>
commonly used to generate efficient chromosomal rearrange-
ments in eukaryotic cell lines and whole organisms (18) but is
rarely used to promote in vivo recombination in bacteria (2,
41). In *L. lactis*, the highest recombination efficiency was ob-
tained without induction of *cre* and in the absence of selective
pressure. This indicates that the basal level of Cre recombinase
in the cell, caused by a low level of leakiness of the *nisA*
promoter, is sufficient to promote efficient recombination be-
tween the *loxP* sites, suggesting that very few Cre protein
molecules are necessary in the cell to allow the full recombi-
nation process. The decrease of the inversion efficiency be-
tween the two distal *loxP* sites observed after overexpression of
the *cre* gene by nisin induction can be explained in light of a
recent study which demonstrated that in *E. coli*, overexpression
of the Cre recombinase altered the directionality for recombi-
nation, leading to deletion instead of inversion in substrates
carrying two *loxP* sites as inverted repeats (3).

To demonstrate the usefulness of the Cre-*loxP* recombina-
tion system, three large chromosomal inversions have been
constructed at different locations in the genome of *L. lactis*. The
first inversion (interval, 307/1-*pcp*) changes the orientation of
one half of the right replication arm without modifying the
chromosome symmetry with respect to *oriC*. The second inver-
sion (interval, 307/1-*ilvD*) results in strong asymmetrical chro-
mosome replication: the “lower” (counterclockwise-moving)
fork replicates about 92% of the chromosome and meets the
opposite fork 180 kb from *oriC*. The last inversion (interval,
307/5-*pcp*) does not alter the length of the two replication arms
or the location or orientation of genes with respect to *oriC*. All
inversions have an effect on the cell fitness compared to that
associated with the corresponding isogenic parental structure,
with a decrease in growth rate ranging from 8 to 18% depend-
ing on the extent of the chromosome disorganization. Exper-
imentation involving additional large-scale inversions with
endpoints located at various sites on the chromosome and
investigation of the genetic behavior of these rearrangements
(by monitoring their stability) as well as of their phenotypic
consequences for cell fitness (by directly comparing the growth
rates of isogenic parental- and inverted-type strains in different
culture conditions) should provide data on the constraints act-
ing to preserve the lactococcal genome organization.

The Cre-*loxP* recombination system has several major ad-
vantages over the other strategies used to generate genome
rearrangements in bacteria. First, the recombination efficiency
is high enough for an easy recovery of rearranged clones with-
out any selection system and seemed to be independent of the
nature of the chromosomal DNA to be inverted. Thus, it can
potentially be used for generating rearrangements in any re-
region of the bacterial chromosome. Second, the efficiency of
Cre-mediated recombination does not depend on the length of
the DNA fragment located between the two inverted *loxP* sites,
because 500-, 1,115-, and 1,160-kb inversions were easily ob-
tained. These results contrast with those found for eukaryotic
cells, in which the recombination frequencies decrease as the
genetic distance between the two *loxP* sites increases (42).
Third, the Cre recombinase does not need any host factors or
additional processes for catalyzing the complete recombina-
tion between the *loxP* sites, in contrast to what has been ob-
served for *S. enterica* serovar Typhimurium, in which inversions
generated by a homologous recombination mechanism require

the RecA and RecBCD proteins (36) and thus may proceed
through extensive DNA replication (19).

In conclusion, the Cre-*loxP* recombination system described
in this paper appears to be a powerful tool for purposes such
as control of the copy number of integrated exogenous DNA in
gene expression investigations or shuffling of the bacterial
chromosome (by deletions or inversions) for applied and fun-
damental genome studies. This recombination system can po-
tentially be used in other gram-positive species without further
modifications, since all of the components described here (se-
lectable markers, ISS1 insertion sequence, and pGhost repli-
con) are functional in alternative gram-positive bacteria (9,
31).

ACKNOWLEDGMENTS

This work was supported by grants from the Centre National de la
Recherche Scientifique (UMR5100), the Region Midi-Pyrénées
(RECH 97002182), and the EC BIOTECH program (BIO4-CT96-
0498). N. Campo was supported by a fellowship from the Ministère de
l'Enseignement Supérieur et de la Recherche.

We thank M. Dias, A. Edelman, and M. J. Pillaire for helping to
improve the manuscript.

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# Chromosomal constraints in Gram-positive bacteria revealed by artificial inversions

N. Campo,† M. J. Dias,† M.-L. Daveran-Mingot, P. Ritzenthaler* and P. Le Bourgeois‡

*Laboratoire de Microbiologie et Génétique Moléculaire du CNRS (UMR5100), Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse cedex, France.*

## Summary

We used artificial chromosome inversions to investigate the chromosomal constraints that preserve genome organization in the Gram-positive bacterium *Lactococcus lactis*. Large inversions, 80–1260 kb in length, disturbing the symmetry of the origin and terminus of the replication axis to various extents, were constructed using the site-specific Cre-LoxP recombination system. These inversions were all mechanistically feasible and fell into various classes according to stability and effect on cell fitness. The *L. lactis* chromosome supports only to some extent unbalance in length of its replication arms. The location of detrimental inversions allowed identification of two constrained chromosomal regions: a large domain covering one fifth of the genome that encompasses the origin of replication (Ori domain), and a smaller domain located at the opposite of the chromosome (Ter domain).

## Introduction

Comparative genomics has significantly increased our understanding of bacterial evolution and has led to the view that bacterial genomes are not static but are instead dynamic, constantly evolving structures (Casjens, 1998; Cole and Saint Girons, 1999), with genome plasticity now regarded as one of the key elements in genome evolution (Watanabe et al., 1997; Mira et al., 2002). Pairwise comparison of closely related bacteria at particular gene positions or DNA motifs has revealed a general X-shaped pattern across an axis defined by the replication origin (*oriC*) and terminus (*terC*) sites (Eisen et al., 2000; Tillier and Collins, 2000; Da Silva et al., 2002; Zivanovic et al., 2002). This suggests that genome rearrangements occur

mainly by a replication-directed recombination process (Bi and Liu, 1996) that generates insertions/deletions/ translocations (Tillier and Collins, 2000) or inversions (Eisen et al., 2000). In addition, most spontaneous inversions include either *oriC* or *terC* regions, do not destroy chromosome symmetry (Hill and Harnish, 1981; Jarvis et al., 1990; Komoda et al., 1991; Römling et al., 1997; Daveran-Mingot et al., 1998; Stibitz and Yang, 1999; Read et al., 2000), and can compensate for DNA insertions altering the relative positions of genes with respect to *oriC* (Liu and Sanderson, 1995a,b). Thus, large inversions, which were initially considered to be rare in bacteria (Roth et al., 1996), are now emerging as one of the major forces driving genome evolution (Hughes, 2000; Kresse et al., 2003).

Although they seem to operate to various extents in the dynamic state of the bacterial chromosome (Casjens, 1998), there is a body of evidence to suggest that physical constraints or selective pressures maintain the global architecture of the chromosome, such as preservation of nearly equal length of the two replication arms (Bergthorsson and Ochman, 1998), conservation of the genetic organization around *oriC* (Smith et al., 1991) and the relative positions of a significant number of orthologous genes with respect to *oriC* (Horimoto et al., 2001). These constraints or pressures also maintain the preferential orientation of highly expressed genes toward *terC* (McLean et al., 1998), or the asymmetric location and the biased orientation of ribosomal operons (Cole and Saint Girons, 1994; Watanabe et al., 1997). Moreover, large-scale sequencing data have revealed GC and oligonucleotide skews between the leading and lagging strands (Lobry, 1996; Mrazek and Karlin, 1998; Salzberg et al., 1998), with a switch in polarity close to the *oriC* and *terC* sites (Grigoriev, 1998; McLean et al., 1998). Compositional asymmetry has also been observed at the gene level, with codon usage or amino acid composition of the encoded protein depending on whether the gene is located on the leading or the lagging strand (Rocha et al., 1999). All these skews are thought to reveal or contribute to the maintenance of the bacterial genome organization. If conceivably some evolutionary processes act to optimize the genome organization in order to allow the best adaptive response of the cell within its natural environment (Rocha et al., 2000), the diversity in the size and shape of genomes encountered in prokaryotes raises the

<footnote>Accepted 24 September, 2003. *For correspondence. E-mail ritzenth@ibcg.biotoul.fr; Tel. (+33) 561 335825; Fax (+33) 561 335886. †These authors contributed equally to the work. ‡Dedicated to Lina Le Bourgeois (10/02/2002).</footnote>
possibility that chromosomal constraints differ according
to the lifestyle of the bacterium.

Large-scale aspects of chromosome organization have
been systematically analysed only in *Escherichia coli* and
*Salmonella enterica* sv Typhimurium, by experimentally
constructing inversions. Despite the close phylogenetic
relationship between these two species, these studies led
to divergent interpretations concerning the behaviour of
the inversions. In *E. coli*, two large regions (NDZ for Non-
Divisible Zones) located near the replication terminus
have been found to be refractory to inversions (Rebollo
et al., 1988; Guijo et al., 2001). It has been hypothesized
that all the inversions tested were mechanistically feasible
but that those ending in the NDZs had deleterious or lethal
effects (functional hypothesis), suggesting a role for NDZs
in nucleoid organization and/or chromosome segregation.
In contrast, no such NDZ region was identified in the *S.
enterica* sv. Typhimurium chromosome (Segall et al.,
1988; Krug et al., 1994). In this case, it was suggested
that the failure to generate inversions in particular zones
was due mostly to a failure of the intrachromosomal
recombination process as a whole rather than to detri-
mental effects on cell fitness (mechanistic hypothesis)
(Mahan and Roth, 1991; Miesel et al., 1994).

In terms of evolution, it is essential to determine exper-
imentally if constraints on long-range chromosome
organization operate similarly in bacterial species phylo-
genetically and/or ecologically unrelated to these two
enterobacteria. *Lactococcus lactis* is a low G + C Firmic-
ute that belongs to the *Streptococcaceae* family and has
numerous health and dairy applications. Complete
genome sequencing revealed a structure characteristic of
eubacteria (Bolotin *et al.*, 2001): the lactococcal chromo-
some is divided into two replication arms almost equal in
length (47 and 53% of the genome, respectively), with
each arm displaying strong bias in gene orientation, GC
skew, oligonucleotide skew (P. Le Bourgeois, unpublished)
and Chi site orientation (el Karoui *et al.*, 1999) with
respect to the direction of replication.

We report here the characterization of two regions at
opposite ends of the lactococcal chromosome that were
refractory to inversions generated by a site-specific
recombination system. We also show that all inversions
were mechanistically possible but that some were highly
unstable and detrimental to cell fitness or led to a cell
death phenotype.

Results

Inversions of the lactococcal chromosome generated by
Cre-mediated recombination

Using a strategy recently described (Campo *et al.*,
2002), we constructed inversions from 18 intervals by

introducing two inverse-order *loxP* sites into the chro-
mosome of *L. lactis* (Fig. 1A). The size of the inverted
segment ranged from 80 to 1260 kb (Table 1). One
endpoint of each interval was arbitrarily fixed at the
midpoint of the left replication arm by inserting one of the
*loxP* sites into the *pcp* region *via* single cross-over
(SCO), whereas the second *loxP* site was integrated
randomly *via* replicative transposition of the lactococcal
IS element *ISS1*. As SCO or transpositional integration
of exogenous DNA in *L. lactis* generally leads to
amplification of the integrated DNA, regardless of the
selective marker used, each double integrant carried
multiple copies of the integrative plasmids (data not
shown). Recombination between the various copies of
the *loxP* sites was assessed in each double integrant,
after introducing the Cre recombinase encoded on a
temperature-sensitive replication plasmid (pGh-Cre), as
described in Fig. 2. In conditions permissive for pGh-
Cre replication, the Cre recombinase catalysed recom-
bination between the various *loxP* sites and generated
two types of chromosomal rearrangement: deletion of
the integrated multicopy plasmids by recombination
between direct-order *loxP* sites (*in vivo* internal control
of Cre activity), and inversion of the DNA fragment
bordered by two inverse-order *loxP* sites. The revers-
ibility of Cre recombination theoretically leads to equi-
librium between parental- and inverted-type genomes
in the bacterial population. Depending on the interval
studied, the percentage of inverted-type chromosomes
ranged from 6 to 58% of the cell population (Table 1,
and Fig. 3), and this recombination efficiency was not
correlated to the length of the inverted chromosome
fragment (Spearman's coefficient = 0.1, *P* = 0.7). This
result contrasts with those found in eukaryotic cells, in
which the Cre-mediated recombination frequencies
decreases as the genetic distance between the two
*loxP* sites increases (Zheng *et al.*, 2000). Removal of
the Cre-encoding plasmid in temperature-shift experi-
ments made it possible to isolate pairs of isogenic
strains containing either the parental or the inverted-
type chromosome in 14 of the 18 intervals tested.
Pulsed field gel electrophoresis (PFGE) macro restric-
tion pattern analyses of the structure of each chromo-
some revealed no rearrangement other than the
predicted inversion (data not shown). No inverted-type
strain could be isolated from the intervals [pcp-20],
[pcp-11], [pcp-37] and [pcp-36]. However, PFGE analy-
sis showed that the fragment indicating the presence
of the Cre-mediated inversion was present in each of
the four strains carrying pGh-Cre (Fig. 3 LEFT, lane 4
and Fig. 3 RIGHT). This result strongly suggests that
the failure to obtain inverted-type strains from these
intervals was caused by a lethal phenotype of the
inversion, rather than a mechanistic problem.
<figure><img src="image_80.png" /><figcaption>**Fig. 1.** A. Location of the *loxP* sites used to generate the various inversions in the *L. lactis* NZ9000 chromosome. The fixed endpoint of all inversions is located in the *pcp* region, whereas the variable endpoint is numbered. An example of three inversions is indicated with the corresponding labelling used in text. As strain NZ9000 is a derivative of the laboratory strain MG1363, and as no macrorestriction polymorphism was observed by PFGE, after digestions with *Apal*, *SmaI*, *NotI*, or *I-Ceul*, between the chromosomes of the two strains (data not shown), the physical map of the MG1363 chromosome (Le Bourgeois *et al.*, 1995) was used as a reference. Black triangles indicate the ribosomal operons (*rrnA* to *F*). The *oriC* region was defined based on its location on the sequenced *L. lactis* IL1403 chromosome (Bolotin *et al.*, 2001). The *dif* site, located at the region that switches in GC and oligonucleotide skews, was deduced from experimental results (Le Bourgeois P., unpubl. results). B. Chromosomal location of the different classes of inversions. Constrained chromosomal regions (corresponding to class III and IV inversions) are indicated in grey. Dotted line indicates the right limit of the putative 'extended Ter domain'.</figcaption></figure>

<table><caption>**Table 1.** Behaviour of the inversions.</caption><thead><tr><th rowspan="2">Interval</th><th rowspan="2">Size of the<br>inversion<br>(in kb)</th><th rowspan="2">% of<br>chromosome<br>inverted<sup>a</sup></th><th colspan="2">Stability</th><th rowspan="2">Class</th><th colspan="2">α μ<sub>A</sub>/μ<sub>B</sub></th></tr><tr><th>GM17</th><th>MS10</th><th>GM17</th><th>MS10</th></tr></thead><tbody><tr><td>[pcp-30]</td><td>80</td><td>45</td><td>S</td><td>S</td><td>I</td><td>1.01 ± 0.02<sup>b</sup></td><td>0.93 ± 0.03</td></tr><tr><td>[pcp-26]</td><td>380</td><td>41</td><td>U<sup>c</sup></td><td>S</td><td>II</td><td>1.06 ± 0.11</td><td>1.03 ± 0.02</td></tr><tr><td>[pcp-1]</td><td>500</td><td>15</td><td>U</td><td>U</td><td>III</td><td>1.17 ± 0.05</td><td>0.94 ± 0.11</td></tr><tr><td>[pcp-20]</td><td>570</td><td>19</td><td>NA</td><td>NA</td><td>IV</td><td>NA</td><td>NA</td></tr><tr><td>[pcp-6]</td><td>860</td><td>15</td><td>U</td><td>U</td><td>III</td><td>1.34 ± 0.13</td><td>0.96 ± 0.09</td></tr><tr><td>[pcp-31]</td><td>870</td><td>31</td><td>U</td><td>S</td><td>II</td><td>1.31 ± 0.16</td><td>0.96 ± 0.00</td></tr><tr><td>[pcp-12]</td><td>1140</td><td>44</td><td>S</td><td>S</td><td>I</td><td>1.03 ± 0.12</td><td>0.93 ± 0.03</td></tr><tr><td>[pcp-34]</td><td>1260</td><td>45</td><td>S</td><td>S</td><td>I</td><td>0.96 ± 0.03</td><td>0.97 ± 0.03</td></tr><tr><td>[pcp-5]</td><td>1160</td><td>50</td><td>S</td><td>S</td><td>I</td><td>1.08 ± 0.09</td><td>1.06 ± 0.04</td></tr><tr><td>[pcp-3]</td><td>1040</td><td>49</td><td>S</td><td>S</td><td>I</td><td>1.00 ± 0.03</td><td>0.99 ± 0.01</td></tr><tr><td>[pcp-13]</td><td>835</td><td>58</td><td>S</td><td>S</td><td>I</td><td>0.99 ± 0.04</td><td>0.94 ± 0.03</td></tr><tr><td>[pcp-10]</td><td>615</td><td>22</td><td>U</td><td>S</td><td>II</td><td>1.02 ± 0.04</td><td>0.97 ± 0.02</td></tr><tr><td>[pcp-40]</td><td>605</td><td>15</td><td>U</td><td>S</td><td>II</td><td>1.22 ± 0.10</td><td>1.03 ± 0.06</td></tr><tr><td>[pcp-36]</td><td>570</td><td>12</td><td>NA</td><td>NA</td><td>IV</td><td>NA</td><td>NA</td></tr><tr><td>[pcp-37]</td><td>530</td><td>6</td><td>NA</td><td>NA</td><td>IV</td><td>NA</td><td>NA</td></tr><tr><td>[pcp-11]</td><td>500</td><td>10</td><td>NA</td><td>NA</td><td>IV</td><td>NA</td><td>NA</td></tr><tr><td>[pcp-35]</td><td>370</td><td>34</td><td>S</td><td>S</td><td>I</td><td>1.07 ± 0.04</td><td>1.07 ± 0.05</td></tr><tr><td>[pcp-4]</td><td>140</td><td>30</td><td>U</td><td>U</td><td>III</td><td>1.10 ± 0.04</td><td>1.02 ± 0.03</td></tr></tbody></table>

a. Calculated as the proportion of inverted chromosomes in the Cre-containing cell population grown in GM17 (see *Experimental procedures*).

b. Standard deviation.

c. Reversion is observed only after 80 generations.

μ<sub>A</sub>: μ<sub>max</sub> of parental-type strain.

μ<sub>B</sub>: μ<sub>max</sub> of inverted-type strain.

NA: Not applicable.
<figure><img src="image_81.png" /><figcaption>**Fig. 2.** Theoretical example for the PFGE-visualization of a chromosomal rearrangement in *L. lactis*. The integration of pCL307 and pCL311 into the chromosome provided additional *Not* I (N) or *I-Ceul* (C) restriction sites making it possible to determine their precise location and orientation on the chromosome. In the double integrant (DI), Southern blotting with the *tetL* probe revealed a chromosomal fragment (*X*<sub>C</sub> or *X*<sub>N</sub>) in addition to the 5.4 kb fragment corresponding to the multicopy insertion of the plasmids. Transient production of the Cre recombinase (see *Experimental procedures*) promoted recombination between the various *loxP* sites and made it possible to isolate isogenic strains containing either the parental chromosome structure (*P*, as shown by the detection of the *X*<sub>C</sub> or *X*<sub>N</sub> fragment on hybridization), or the inversion (*I*, as shown by the detection of the *Y*<sub>C</sub> or *Y*<sub>N</sub> fragment on hybridization). Unstable inversion resulted in a reversion to a parental-type chromosome structure by homologous recombination between either the two copies of the *pcp* region (*R*<sub>pcp</sub>, visualized by detection of the *X*<sup>*</sup>*C* or *X*<sup>*</sup>*N* fragment on hybridization, these fragments being very similar in size to the parental *X*<sub>C</sub> and *X*<sub>N</sub> fragments, respectively), or the two copies of *ISS1* (*R*<sub>ISS1</sub>, visualized by detection of two new hybridizing fragments, *Z*<sub>C</sub> or *Z*<sub>N</sub>). The letters a, b, c, and d indicate order on the chromosome.</figcaption></figure>

## Behaviour of the inversions

The 14 inverted-type strains isolated in *L. lactis* were analysed for the stability of the genome (i.e. reversal toward the parental-type chromosome structure) and for their phenotypic consequences on cell growth. We postu-

lated that if there are constraints maintaining the native lactococcal genome organization, then the inverted-type bacteria obtained by Cre recombination would be at a selective disadvantage and should acquire compensatory mutations or rearrangements to restore their original chromosome structure.
<figure><img src="image_82.png" /><figcaption>**Fig. 3.** Efficiency of Cre-mediated recombination. Double integrants containing pGh-Cre were grown in condition permissive for plasmid replication, and their genomes were analysed by PFGE (electrophoresis conditions: 275 V/7 s of pulse time/14 h), after Apal digestion, followed by Southern blotting with tetL as a probe. Arrows indicate fragments characteristic of the inverted-type structure. Left (electrophoresis conditions: 275 V/7 s of pulse time/14 h); Lanes: M, λ concatemer size standards; L, HindIII-digested λ DNA; 1, [pcp-1]; 2, [pcp-6]; 3, [pcp-13]; 4, [pcp-20]; 5, [pcp-31]; 6, [pcp-35]; 7, [pcp-10]; 8, [pcp-5]. Right (electrophoresis conditions: 275 V/2 s pulse time/14 h); Lanes: M, λ concatemer size standards; L, HindIII-digested λ DNA; 1, [pcp-11]; 2, [pcp-36]; 3, [pcp-37]. Hybridizations were performed in the presence of ³²P-labelled λ DNA.</figcaption></figure>

We determined the specific growth rate ($\mu_{max}$) of each pair of isogenic strains carrying parental or inverted chromosomes in various growth media (Table 1). In synthetic medium (MS10), where $\mu_{max}$ is low ($\approx 0.9 h^{-1}$ for the wild-type strain), no significant difference of growth was observed between parental-type and inverted-type strains (Student's test, $t = 1.11, P = 0.28$). In complex medium (GM17), where $\mu_{max}$ is higher ($\approx 1.4 h^{-1}$ for the wild-type strain), inversions fell into two phenotypic classes (Student's test, $t = 7, P < 0.0001$): those that did not change the growth rate of the bacteria (intervals [pcp-3], [pcp-4], [pcp-5], [pcp-10], [pcp-12], [pcp-13], [pcp-26], [pcp-30], [pcp-34]), and [pcp-35], and inversions that conferred a selective disadvantage with respect to the parental structure (intervals [pcp-1], [pcp-6], [pcp-31] and [pcp-40]). Thus, the inversion of some chromosomal regions has a negative effect on growth rate, but only in conditions in which $\mu_{max}$ is the highest. Location of the detrimental inverted intervals on the lactococcal chromosome reveals that the disruption of native genome organization around the origin of replication was of greatest selective disadvantage.

We determined the stability of each inversion over 150 generations of cell growth in complex or synthetic medium by PFGE-analysis of the chromosome structure

in the cell population (Fig. 4). With the genetic system used in this study, it was possible for the inverted chromosome to revert to a parental-type structure in the absence of Cre recombinase, by homologous recombination between the two copies of the 0.8 kb insertion sequence *ISS1* (Fig. 2, $R_{ISS1}$), or the 5 kb *pcp* region (Fig. 2, $R_{pcp}$). In *L. lactis*, recombination frequency varies exponentially with the size of the homologous targets, and decreases by a factor of 100 for targets less than 1 kb in size (Biswas *et al.*, 1993). We therefore thought it likely that the major event accounting for reversion to the parental chromosome structure would be homologous recombination between the duplicated *pcp* fragments. The isolated inversions fell into three stability classes (Table 1): seven inversions were shown to be stable in both media (class I inversions, intervals [pcp-3], [pcp-5], [pcp-12], [pcp-13], [pcp-30], [pcp-34] and [pcp-35]), four inversions were stable in MS10 but not in GM17 (class II inversions, intervals [pcp-10], [pcp-31], [pcp-40], and, to a lesser extent [pcp-26] for which reversion was not observed before 80 generations) and three inversions were unstable in both media (class III inversions, intervals [pcp-1], [pcp-4] and [pcp-6]).

Analysis of the reversion of recombined regions revealed that unstable inversions reverted to the
A

<figure><img src="image_83.png" /></figure>

B

<figure><img src="image_84.png" /><figcaption>**Fig. 4.** Stability of inverted chromosome during serial growth for 150 generations in GM17 (left panels) or MS10 (right panels). The genome of inverted-type strains was analysed by I-Ceul digestion and PFGE, followed by Southern blotting with tetL as the probe.</figcaption></figure>

A. Example of an unstable inversion (class III), interval [pcp-1]. Electrophoresis conditions: 275 V/15 s of pulse time/14 h.

B. Example of a stable inversion (class I), interval [pcp-5]. Electrophoresis conditions: 275 V/20 s of pulse time/14 h. The number above each lane indicates generation number. Abbreviation: P, parental-type chromosome; I, inverted-type chromosome; M, λ DNA concatemer size standards. Hybridization was performed in the presence of ³²P-labeled λ DNA.

parental structure almost exclusively by homologous recombination between the two *pcp* fragments (data not shown). However, two inversions (intervals [pcp-4] and [pcp-31]) reverted by recombination at the *pcp* and *ISS1* loci, at similar frequencies (data not shown). Therefore, unexpected high frequency recombination at the *ISS1* loci should reflect local constraints in addition to those associated with the inversion. This is probably the case for interval [pcp-4], which corresponds to an unstable region in the NZ9000 chromosome that contains a transposon-like structure, a cryptic prophage, and the unique endogenous copy of the *ISS1* element (Le Bourgeois *et al*., 2000a).

*Unstable and lethal inversions revealed two constrained domains at opposite ends of the chromosome*

We mapped the inverted intervals on the physical map of the *L. lactis* chromosome. The distribution of the various classes of inversion was not random (Fig. 1B). Most of the well-tolerated inversions (class I) had one endpoint in each arm of the second half of the chromosome, whereas unstable (class II and III) and lethal (class IV) inversions were preferentially positioned in two regions at opposite ends of the chromosome. The first region, the 'Ori domain', is a 500 kb region (corresponding to one fifth of the chromosome) centred on the replication origin and delimited by the endpoints of two class II inversions (26
and 31). The second constrained domain, the 'Ter domain', is 105 kb or longer, extended from endpoint 11 to endpoint 10, and was asymmetrically located close to the putative lactococcal *dif* site (P. Le Bourgeois, unpubl. obs.).

Discussion

Chromosomal inversions are the most appropriate rearrangement for the experimental analysis of genome organization because they disturb the normal order of sequences on the chromosome without affecting overall genetic content. To date, such studies have been carried out only for two closely related bacteria, *E. coli* and *S. enterica* sv Typhimurium, either by reciprocal recombination between two inverse-order homologous sequences (Francois *et al.*, 1987; Mahan and Roth, 1988) or by intramolecular transposition of *Tn10* (Krug *et al.*, 1994). This study reports the first systematic analysis of the constraints maintaining native genome organization in a bacterium unrelated to *Enterobacteriaceae*, by means of artificial inversions of the *L. lactis* chromosome, using the site-specific Cre-*loxP* recombination system (Campo *et al.*, 2002).

The inversions investigated from 18 intervals of the lactococcal chromosome fell into four classes, depending on their behaviour. Class I inversions were stable over 150 generations and did not modify the growth characteristic of the cells, suggesting that no structural (e.g. disruption of large-scale genome organization) or functional (e.g. alteration of gene expression) constraints occur in these regions, whatever the growth conditions. However, it cannot be excluded that these inversions confer a slight selective disadvantage during long-term evolution. Class II inversions were unstable only in complex (rich) medium, where the specific growth rate is maximal. We speculate that the resulting inverted genome structure could interfere with the need of a tight coordination of some cellular processes (e.g. chromosome segregation and cell division) or space-time expression of some genes during fast growth. Class III inversions were highly unstable in every growth condition tested. Mathematical simulation (taking into account specific growth rate of inverted- and parental-type clones and kinetics of instability) suggests that inverted genome structures reverted toward the parental structures at a frequency of 10⁻² per cell and per generation (data not shown). Class IV inversions were so detrimental that they were never isolated.

Large-scale analyses of genome organization in Enterobacteria have shown that some chromosomal regions are refractory to inversion. This finding has been interpreted in two different ways: (i) the mechanistic hypothesis, in which the failure to generate inversions in particular regions is mainly due to failure of the intrachomosomal

homologous recombination process (Miesel *et al.*, 1994), and (ii) the functional hypothesis, in which inversions in non-permissive regions have deleterious effects on cell fitness (Rebollo *et al.*, 1988). Our analysis strongly supports the functional hypothesis since the signature of a full Cre-mediated recombination was identified in every interval tested, even when no viable inverted-type strain could be isolated (class IV inversions). This indicates that no mechanistic barrier exists to prevent Cre-mediated interaction between the *loxP* sites in the *L. lactis* chromosome.

Location of the different classes of inversions on the *L. lactis* chromosome provides some experimental clues on constraints acting on its organization, at least in laboratory growth conditions. Although it cannot be totally ruled out that detrimental effect of inversions is generated by local constraints at the *pcp* locus used as the fixed endpoint, we consider this is not the case as half (7/14) of the inversions constructed in this study are stable over 150 generations and have no significant effect on the cell growth. The lactococcal chromosome is able to support variation of the *oriC/terC* symmetry (with *terC* located at the switch of GC and oligonucleotide skews and corresponding to the putative *dif* site), but apparently only to some extent. Inversions that modified the relative length of the two replication arms by 2% (intervals [pcp-5] and [pcp-34]), 7% (intervals [pcp-12] and [pcp-3]), or 15% (interval [pcp-13]) are stable and have no effect on cell growth, whereas inversions displacing the *terC* site by 22 or 23% (intervals [pcp-10] and [pcp-40], respectively) are unstable in complex medium (GM17). Similar results have been previously found in *E. coli*, where inversions that maintain the *oriC/terC* symmetry did not change the growth rate of the cells (Hill and Gray, 1988), whereas those altering this symmetry reduced the cell fitness and led to secondary inversions that restored the wild-type chromosomal structure (Louarn *et al.*, 1985; Hill and Gray, 1988).

Large spontaneous inversions in the same replication arm, which maintain the *oriC/terC* symmetry but reverse the genes orientation, have not been observed by comparative genomics of closely related chromosomes, suggesting a selective disadvantage for such rearrangements in natural conditions. However, this kind of inversions can be easily recovered in laboratory conditions (Rebollo *et al.*, 1988). In *L. lactis*, inversions in the same replication arm are also easily obtained and lead to different behaviours, depending of the location of the second endpoint. Three inversions are stable (intervals [pcp-30] and [pcp-35]) or slightly unstable (interval [pcp-26]) and have no effect on cell growth, whereas other inversions (intervals [pcp-1], [pcp-20], [pcp-11], [pcp-37], and [pcp-36]) belong to Class III and Class IV. It has been observed that low G + C Firmicutes possess a gene strand bias greater than
other taxonomic groups. This could be related with the asymmetry in the replication fork machinery (Rocha, 2002). Then, one might argue than the strong detrimental effect observed when inverting large segments of the same replicore is mainly due to the displacement of the leading strand genes to the lagging strand, especially if these genes are essential (Rocha and Danchin, 2003). Although our experimental data seem to correlate well with this assertion, the patchwork of the different classes of inversions and the location of endpoints close to *oriC* and *terC* regions for the most detrimental inversions support the hypothesis that additional constraints exist in the lactococcal genome organization. For example, location of the endpoints of the three lethal inversions (intervals [pcp-11], [pcp-36], and [pcp-37]) near *terC* is in good agreement with the polarization of the replicores associated with the regional control of the recombination at the chromosomal *dif* site in *E. coli* (Perals *et al.*, 2000): inversions that do not encompass the *dif* site disrupt the polar elements necessary for the correct Xer recombination and lead to a lethal phenotype, whereas inversions that include the *dif* site (i.e. intervals [pcp-40], and [pcp-10]) are less detrimental. As occurrence of polar elements seems a general feature in bacterial genomes (Salzberg *et al.*, 1998), we postulate that inversions symmetrical to intervals [pcp-11], [pcp-36], and [pcp-37] should confer a lethal phenotype. Thus, the lactococcal 'extended Ter domain' should be a 250-kb region centred on the putative *dif* site (Fig. 1B). However, further inversions in this chromosomal region are required to confirm this hypothesis. We speculate that if constraint at the terminus is a common feature of the bacterial chromosome, intrinsic organization might differ between species. In *E. coli*, the 'Ter domain' is a 900 kb region (Niki *et al.*, 2000) containing two distinct regions refractory to inversion (NDZ1 and NDZ2) that are involved in nucleoid folding/segregation (Guijo *et al.*, 2001). These NDZs are centred on a third region (DAZ, for Dif Activity Zone), involved in chromosome dimer resolution (Cornet *et al.*, 1996). In *L. lactis*, which contains a chromosome half the size of that of *E. coli*, the 'Ter domain' may consist of only one 250 kb continuous region involved in all three functions (i.e. folding/segregation, decatenation and dimer resolution of the chromosome). This possibility is currently under investigation. However, we cannot rule out the possibility that the highly detrimental phenotype of inversions in the 'Ter domain' is due to reversal of the replication terminus sites (*Ter* sites) (Mahan and Roth, 1991), although no equivalent system of the *E. coli* Tus/*Ter* or the *B. subtilis* RTP/*Ter* has been identified in most of the sequenced bacterial genomes.

Opposite to the 'Ter domain', we identified another constrained region in the lactococcal chromosome that surrounds the replication origin. The 'Ori domain' is a large

region defined by five detrimental inversions (intervals [pcp-26], [pcp-1], [pcp-20], [pcp-6], and [pcp-31]), covering about one fifth the length of the chromosome, and is centred on the *oriC* site. Although no such domain has been characterized in Enterobacteria by experimental inversions, the lactococcal 'Ori domain' seems to be similar to the *E. coli* 'Ori domain' defined by FISH (fluorescence *in situ* hybridization) experiments (Niki *et al.*, 2000). The lactococcal 'Ori domain' contains four of the six ribosomal operons (*rrnA* to *D*), a large cluster containing 25 of the 55 genes encoding ribosomal proteins (the S10 operon), and at least seven predicted highly expressed genes (PHX genes) (Karlin *et al.*, 2001). It has been suggested that changes in gene dosage may account for the growth defects of some inverted strains in *S. enterica* sv Typhimurium (Segall *et al.*, 1988). As the doubling time of *Lactococcus* in complex medium (GM17) is very similar to the chromosomal replication time of *Streptococci* (Coper, 1991), suggesting that the maximum origin/terminus ratio should be 2 in *L. lactis* cells, such a gene dosage effect is unlikely to account fully for the features of the 'Ori domain'. However, we cannot exclude the possibility that displacement of the S10 operon or another PHX gene away from *oriC* has phenotypic consequences, even if the gene dosage effect is only weak. It is also possible that inversions separate two genes or gene clusters, reducing the efficiency of coordinated expression of these genes in growing cells. An alternative explanation would be that inversions disrupting the structure of the 'Ori' region could have a negative effect on the chromosome segregation process, as there are evidences for existence of particular *cis*-acting sequences located in the vicinity of *oriC* and essential for correct chromosome partitioning. These elements include the *parS* elements in *Streptomyces coeli-color* (Jakimowicz *et al.*, 2002) and *Pseudomonas putida* (Godfrin-Estevenon *et al.*, 2002), uncharacterized centromere-like sequences in *E. coli* (Gordon *et al.*, 2002), or the polar localization region (PLR) recently identified by artificial chromosome inversions in *B. subtilis* (Wu and Errington, 2002).

The main conclusion of this study is that all chromosomal inversions are mechanistically feasible with a site-specific recombination system, that the lactococcal genome support only to some extent unbalance in the length of its replication arms, and that detrimental inversions are mostly located in two opposite regions close to the *oriC* and *dif* sites.

## Experimental procedures

### Plasmids, bacterial strains and growth conditions

The plasmids and bacterial strains used in this study are listed in Table 2. *Lactococcus lactis* strains were grown semi-anaerobically at 30°C in M17 broth (Merck kgaA,
Darmstadt, Germany) supplemented with 0.5% (wt/vol) glucose (GM17 medium), or in MS10 medium (Cocaign-Bousquet *et al.*, 1995) supplemented with thiamine (1 mg l⁻¹) and lipoic acid (2.5 mg l⁻¹). Antibiotics were used at the following concentrations: erythromycin (Em) 5 µg ml⁻¹; chloramphenicol (Cm) 5 µg ml⁻¹, and spectinomycin (Spc) 200 µg ml⁻¹. Growth rates were determined in GM17 or MS10 broth as follows. Culture tubes (diameter, 16 mm) containing 5 ml of prewarmed medium were inoculated with 2% of an overnight culture and incubated at 30°C, without shaking, in a water bath. Bacterial growth was monitored by spectrophotometric measurements of optical density (OD) at 600 nm (model UV-1205; Shimadzu, Kyoto, Japan) every 30 min until the culture reached stationary phase. The growth curve of each strain was deduced from six to nine independent experiments.

## DNA manipulation

Restriction enzymes were purchased from New England Biolabs (Beverly, MA) and used as recommended by the supplier. *Lactococcus lactis* strains were electrotransformed as previously described (Le Bourgeois *et al.*, 2000b) with 5 µg of integrative plasmid or 50 ng of the replicative plasmid pGh-Cre. Plasmid DNA from *E. coli* was isolated with the 'Qiaprep spin' kit (small scale) or 'Qiagen plasmid Midi' kit (large-scale) according to the manufacturer's instructions (Qiagen GmbH, Hilden, Germany). Preparation of lactococcal genomic DNA embedded in agarose matrix, PFGE, and Southern blotting with dried agarose gels were performed as previously described (Le Bourgeois *et al.*, 1995). Hybridization signals were detected with a Bioimaging BAS1000 analyzer system (FUJI Photo Film, Tokyo, Japan) and analysed with TINA version 2.07c software (Raytest Isotopenβgeräte GmbH, Straubenhardt, Germany).

## Isolation of chromosomal inversions

A detailed description of the protocols and plasmids used for the Cre-*loxP* recombination system from coliphage P1

(Abremski *et al.*, 1983) in *L. lactis* has been published elsewhere (Campo *et al.*, 2002). Strain NZ9000 (Table 2) was transformed with the transpositional plasmid pCL307 and integrants were selected for growth on Em. As all the integrative plasmids used in this study contained single *Apal*, *Smal*, *NotI*, and *I-Ceul* restriction sites, the sites and orientation of integrations into the chromosome of lactococcal strains were determined by Southern blotting of PFGE-separated chromosomal restriction fragments, as previously described (Le Bourgeois *et al.*, 1992). Several clones were chosen for consecutive transformation with pCL311 or pCL322, depending on the chromosomal orientation of pCL307, and integrants were selected for growth on Spc. The plasmids pCL311 and pCL322 integrate into the *pcp* region by homologous recombination and differ only in the orientation of the lactococcal *pcp* region. Each double integrant (DI, Fig. 2) was transformed with the temperature-sensitive replication plasmid pGh-Cre, and cells were selected for growth on Cm. The genomic structure of the selected cells was analysed by PFGE and Southern blotting, using the *tetL* ORF as probe, and Cre-mediated inversion efficiency was quantified by calculating the ratio of the hybridization signal of the signature fragment of the inverted-type chromosome (I, Fig. 2) to the sum of the signals of the fragments characterizing the inverted- and parental-type chromosome (I + P, Fig. 2). Strains carrying the parental- or inverted-type chromosome were isolated after eliminating pGh-Cre as follows. Double integrants containing pGh-Cre were cultured overnight in GM17-Cm medium at 28°C, diluted 1:100 in 5 ml of GM17 without antibiotics, and incubated for 2 h at 28°C (water bath) to allow exponential growth to resume. The cultures were then shifted to 37.5°C for 7 h (water bath), and 50 µl of a 10⁻⁵ dilution was plated on GM17 plates and incubated at 28°C. One hundred colonies were transferred with tooth-picks to non-selective and chloramphenicol-containing GM17 plates and incubated at 28°C. The genomic structure (parental-type P or inverted I, Fig. 2) of the Cm⁵ colonies was determined by PFGE and Southern blotting with the *tetL* ORF probe, and the absence of pGh-Cre was confirmed by Southern blotting, using the *cre* gene as a probe.

<table><caption>**Table 2.** Bacterial strains and plasmids used in this study.</caption><thead><tr><th>Plasmid or strain</th><th>Relevant properties</th><th>Origin</th></tr></thead><tbody><tr><td><strong>Plasmids</strong></td><td></td><td></td></tr><tr><td>pCL307</td><td>Em<sup>r</sup>, pCL60 with loxP2 cassette inserted into the EcoRI site</td><td>Campo <i>et al.</i> (2002)</td></tr><tr><td>pCL311</td><td>Spc<sup>r</sup>, pCL302 with a 4.8 kb EcoRI-HindIII fragment of pcp region inserted between the EcoRI and HindIII sites</td><td>Campo <i>et al.</i> (2002)</td></tr><tr><td>PCL322</td><td>Spc<sup>r</sup>, pCL302 with a 6 kb HindIII fragment of the pcp region inserted into the HindIII site in the opposite orientation to pCL311</td><td>This work</td></tr><tr><td>pGh-Cre</td><td>Cm<sup>r</sup>, temperature-sensitive replication plasmid carrying the cre gene downstream from an inducible lactococcal promoter</td><td>Campo <i>et al.</i> (2002)</td></tr><tr><td><strong>Strains</strong></td><td></td><td></td></tr><tr><td><em>Lactococcus lactis</em></td><td></td><td></td></tr><tr><td>MG1363</td><td>Plasmid-free strain</td><td>Gasson (1983)</td></tr><tr><td>NZ9000</td><td>MG1363 pepN::nisRK</td><td>Kuipers <i>et al.</i> (1998)</td></tr><tr><td><em>E. coli</em></td><td></td><td></td></tr><tr><td>DH5α</td><td>F<sup>-</sup> φ80 dLacZΔM15 Δ(lacZYA - argF)U169 endA1 recA1 hsdR17(r<sub>K</sub>-m<sub>K</sub>+) deoR thi -1 supE44 λ<sup>-</sup> gyrA96 relA1</td><td>Invitrogen</td></tr><tr><td>EC1000</td><td>MC1000 glgB::repA</td><td>(Leenhouts <i>et al.</i>, 1996)</td></tr></tbody></table>
Stability analysis of the inversions for 150 generations

Overnight cultures of strains carrying the inverted chromosomal structure were diluted (1:1000) with 10 ml of GM17 or MS10 medium and incubated for 24 h at 30°C. Dilutions and cultures were repeated each day for 15 days. Every day, the bacteria went through a lag phase followed by a period of sustained growth, and a stationary phase, which continued until the next serial transfer. The 1:1000 dilution allowed the bacteria to go through ≈10 cell generations per 24 h. The genomic structure of the cell population ($R_{pcp}$ or $R_{ISS1}$, Fig. 2) was analysed for various generations, by PFGE followed by Southern blotting with the *tetL* ORF probe.

Acknowledgements

This work was supported by grants from the Centre National de la Recherche Scientifique (UMR5100), from the Region Midi-Pyrénées (RECH 97002182), and from the CEC Bio-technology program (BIO4-CT96-0498).

We thank M. J. Pillaire, F. Cornet, and J. Sappa (A. Edelman and associates) for improving the manuscript. P.L.B. is indebted to S. Müller for her help during the final stage of manuscript editing. N. Campo and M. J. Dias were supported by a fellowship from the Ministère de l'Enseignement Supérieur et de la Recherche.

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# Partie 3 : Recombinaison Xer chez les Streptocoques

## Présentation des travaux

Lors de la réplication des chromosomes bactériens circulaires, un événement de recombinaison homologue entre les chromatides sœurs peut conduire à la formation de dimères qui seront incapables de se répartir correctement dans les cellules filles. Chez *E. coli*, la résolution de ces dimères fait intervenir un système de recombinaison spécifique de site, appelé recombinaison Xer (pour **chromosomally encoded recombinase**). Deux recombinases à tyrosine, XerC et XerD, catalysent de façon concertée la recombinaison au site *dif*, un site chromosomique de 28 pb localisé à la jonction des réplichores. Cette recombinaison est intimement couplée à la division cellulaire par l'intermédiaire de FtsK, une protéine bifonctionnelle de 1329 acides aminés de la famille des translocases à ADN FtsK-HerA (lyer *et al.*, 2004), localisée au septum de division et essentielle pour cette dernière fonction (Wang & Lutkenhaus, 1998). FtsK se fixe sur une famille de mots biaisés en orientation sur le chromosome, les KOPS (FtsK **orienting polar sequences**)(Bigot *et al.*, 2005), transloque l'ADN chromosomique de manière unidirectionnelle jusqu'au complexe XerCD-dif (Graham *et al.*, 2010), et active la recombinaison par interaction protéine-protéine avec XerD (Yates *et al.*, 2006). La résolution des dimères de chromosomes est une fonction conservée chez les eubactéries car la majorité des espèces possèdent des orthologues aux gènes *xerC*, *xerD* et *ftsK* d'E. coli (Recchia & Sherratt, 1999), ainsi qu'un site *dif* conservé (Hendrickson & Lawrence, 2007). Ces données suggèrent une origine monophylétique du système Xer. Il peut cependant exister des différences dans le contrôle de la réaction puisque la recombinaison Xer ne semble pas dépendre des homologues de FtsK, SpolIIIE et YtpT (maintenant nommée SftA), chez *B. subtilis* (Sciochetti *et al.*, 2001; Biller & Burkholder, 2009).

Afin de valider le lien entre l'existence de la zone non permissive aux inversions (« domaine Ter », centré sur la région de changement de biais GC du chromosome) et la résolution des dimères de chromosomes, nous avons recherché ce site *dif* sur le chromosome de *L. lactis* et chez différentes espèces de Firmicutes. Nous avons ainsi identifié des sites *dif* fortement similaires au site *dif* de *B. subtilis* chez toutes les espèces testées sauf pour le genre *Streptococcus* et *Lactococcus*. En utilisant une approche de génomique comparative combinée à une validation in vivo chez *L. lactis*, *S. pneumoniae* et *E. coli*, nous avons montré que tous les *Streptococcaceae* possédaient un système de recombinaison Xer atypique et simplifié, constitué d'un site *dif* de 31pb (*dif*<sub>SL</sub>) et d'une recombinase à tyrosine de structure inhabituelle (annotées YmfD chez *L. lactis* et rebaptisée XerS). Ces deux partenaires sont adjacents sur tous les chromosomes analysés et semblent constituer un « module génétique ». Ce système XerS/*dif*<sub>SL</sub> est sous la dépendance de la protéine septale FtsK, mais ne semble pas activé par une interaction protéine-protéine entre FtsK et XerS (**article n°4**).
Depuis ces travaux, une analyse bioinformatique a montré l'existence d'un second système Xer alternatif chez les genres *Helicobacter* et *Campylobacter*, le système XerH/dif<sub>H</sub> (Carnoy & Roten, 2009). Bien que la recombinase XerH semble phylogénétiquement éloignée de XerS (Fig. 10a), le site dif<sub>H</sub> est très similaire à dif<sub>SL</sub> (Fig. 10b) et XerH/dif<sub>H</sub> est organisé en un module génétique comme chez les streptocoques.

<figure><img src="image_85.png" /><figcaption>**Fig. 10.** a) Analyse phylogénétique des recombinases Xer. b) Comparaison des sites *dif*<sub>H</sub> et *dif*<sub>SL</sub>. (d'après (Carnoy & Roten, 2009).</figcaption></figure>

Très récemment, deux études ont démontré expérimentalement l'existence d'un système de résolution des dimères de chromosomes atypique chez plusieurs espèces d'archéobactéries, notamment *Pyrococcus abyssi* (Cortez et al., 2010) et *Sulfolobus solfataricus* (Duggin et al., 2010). Dans ce cas, une seule recombinase (XerA) catalyse la réaction à un site *dif* non conservé entre *S. solfataricus* et *P. abyssi* (Fig. 11).

<figure><img src="image_86.png" /><figcaption>**Fig. 11.** Absence de conservation des sites *dif* chez les archéobactéries. Haut, *S. solfataricus*. Bas, *P. abyssi*.</figcaption></figure>
Nous avons poursuivi l'étude du système XerS/dif<sub>SL</sub> par une caractérisation moléculaire de la machinerie de recombinaison, notamment la structure du site dif<sub>SL</sub>, et le contrôle de la réaction par la protéine FtsK<sub>SL</sub> (article n°5).

Nous avons ainsi montré que XerS se fixe de manière asymétrique et coopérative sur le site dif<sub>SL</sub>, la recombinase possédant une meilleure affinité pour le bras gauche que pour le bras droit, ce qui suggère un ordre d'assemblage défini sur le site de recombinaison. Les réactions de coupure et d'échange de brins se font également de manière asymétrique, puisqu'une jonction de Holliday synthétique est préférentiellement résolue par l'échange des brins inférieurs. Ainsi, bien que la machinerie de recombinaison soit différente, on retrouve des similitudes avec le système XerCD/dif<sub>EC</sub> dans le mode de fixation au site et dans la résolution de la jonction de Holliday. Ces résultats indiquent que l'asymétrie du site dif<sub>SL</sub> est suffisante pour conférer une directionnalité à la réaction de recombinaison.

Chez *L. lactis* comme chez *E. coli*, la résolution des dimères de chromosomes est sous la dépendance de la protéine FtsK. Par l'utilisation de différents allèles du gène *ftsK* (*ftsK*<sub>WT</sub>, *ftsK*<sub>ΔC</sub>, *ftsK*<sub>ΔY</sub>, *ftsK*<sub>KOPSBind</sub> et *ftsK*<sub>ATP-</sub>) nous avons montré que l'activité de translocation de l'ADN était nécessaire, et que FtsK doit atteindre le site *dif*<sub>SL</sub> pour que la réaction de recombinaison soit productive. D'autre part, une protéine FtsK chimérique couplant le domaine C-ter de *L. lactis* aux domaines N-ter et linker d'E. coli (FtsK<sub>CLI</sub>) est capable d'activer les systèmes XerS/dif<sub>SL</sub> et XerCD/dif<sub>EC</sub>. Ces résultats suggèrent donc fortement que les contrôles exercés sur les deux systèmes sont identiques. Cependant la faible conservation sur XerS et FtsK<sub>SL</sub>, des acides aminés considérés comme importants pour l'activation de XerD par le sous-domaine γ de FtsK<sub>EC</sub>, nous a amené à étudier plus en détail ce contrôle de la réaction de recombinaison. Pour cela, nous avons dissocié l'activité de translocation de l'ADN (portée par les sous-domaines α et β) de celle de l'activation par interaction directe avec la recombinase, en produisant les sous-domaines γ de FtsK<sub>SL</sub> et FtsK<sub>EC</sub>. Les résultats obtenus montrent que le sous-domaine γ de FtsK<sub>EC</sub> active la recombinaison XerCD/dif<sub>EC</sub> alors que le sous-domaine γ de FtsK<sub>SL</sub> n'active aucun des deux systèmes. Ces résultats confirment notre hypothèse première que, contrairement à *E. coli*, le contrôle de la recombinaison XerS/dif<sub>SL</sub> ne nécessite pas d'interaction directe entre le sous-domaine γ de FtsK<sub>SL</sub> et la recombinase XerS.
# The Unconventional Xer Recombination Machinery of *Streptococci/Lactococci*

Pascal Le Bourgeois¹,*, Marie Bugarel¹, Nathalie Campo², Marie-Line Daveran-Mingot¹, Jessica Labonté¹, Daniel Lanfranchi¹, Thomas Lautier¹, Carine Pagès¹, Paul Ritzenthaler¹

1 Laboratoire de Microbiologie et Génétique Microbienne, CNRS, Université Paul Sabatier, Toulouse, France, 2 Department of Genetics, Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands

Homologous recombination between circular sister chromosomes during DNA replication in bacteria can generate chromosome dimers that must be resolved into monomers prior to cell division. In *Escherichia coli*, dimer resolution is achieved by site-specific recombination, Xer recombination, involving two paralogous tyrosine recombinases, XerC and XerD, and a 28-bp recombination site (*dif*) located at the junction of the two replication arms. Xer recombination is tightly controlled by the septal protein FtsK. XerCD recombinases and FtsK are found on most sequenced eubacterial genomes, suggesting that the Xer recombination system as described in *E. coli* is highly conserved among prokaryotes. We show here that *Streptococci* and *Lactococci* carry an alternative Xer recombination machinery, organized in a single recombination module. This corresponds to an atypical 31-bp recombination site (*dif*<sub>SL</sub>) associated with a dedicated tyrosine recombinase (XerS). In contrast to the *E. coli* Xer system, only a single recombinase is required to recombine *dif*<sub>SL</sub>, suggesting a different mechanism in the recombination process. Despite this important difference, XerS can only perform efficient recombination when *dif*<sub>SL</sub> sites are located on chromosome dimers. Moreover, the XerS/dif<sub>SL</sub> recombination requires the streptococcal protein FtsK<sub>SL</sub>, probably without the need for direct protein-protein interaction, which we demonstrated to be located at the division septum of *Lactococcus lactis*. Acquisition of the XerS recombination module can be considered as a landmark of the separation of *Streptococci/Lactococci* from other firmicutes and support the view that Xer recombination is a conserved cellular function in bacteria, but that can be achieved by functional analogs.

Citation: Le Bourgeois P, Bugarel M, Campo N, Daveran-Mingot ML, Labonté J, et al. (2007) The unconventional Xer recombination machinery of *Streptococci/Lactococci*. PLoS Genet 3(7): e117. doi:10.1371/journal.pgen.0030117

## Introduction

Chromosome replication is a key function in living cells, and any factor that impedes progression of replication forks can result in mutagenesis and genome instability. Several strategies have evolved to rescue replication forks stalled by DNA damage. Most of these depend on homologous recombination pathways but are not necessarily accompanied by strand exchange [1]. However, in cases where replication fork repair does lead to sister chromatid exchange, bacteria with circular chromosome(s) are faced with a potential topological problem because an odd number of crossovers between sister chromatids generates chromosome dimer, which must be converted back to monomers for a correct segregation to daughter cells. In *E. coli*, chromosome dimer formation occurs in 15% of the cell population [2,3], and conversion to monomers is carried out by the Xer site-specific recombination system (for recent reviews see [4,5]). This is composed of two paralogous tyrosine recombinases (integrases), XerC and XerD, which cooperatively catalyze strand exchanges at a 28-bp DNA sequence, the *dif* site, which must be located at the junction of the two replicohores to be functional [3,6–8]. Xer recombination is intimately coupled to cell division [9] through the septal protein FtsK [10–12], a DNA translocase [8] with an essential N-terminal trans-membrane domain involved in its localization at the septum [13], and a C-terminal DNA motor domain involved in positioning and synapsing the two *dif* sites of the chromosome dimer at the division septum [12,14–19] as well as in

activating the strand exchange [8] by direct interaction with XerD [20,21].

Homologs of XerCD recombinases and FtsK are found in most eubacterial phyla and some archeal lineages [22] as well as the canonical *dif* site [23]. Moreover, interactions between the *E. coli* *dif* site and the XerCD recombinases of *Haemophilus influenzae* [24], *Pseudomonas aeruginosa* [25], *Bacillus subtilis* [26], *Proteus mirabilis* [27], and *Caulobacter crescentus* [28] have been experimentally demonstrated in vitro. These observations led to the general view that Xer recombination is a function conserved among bacteria harboring circular chromosome(s). However, regulation of strand exchange may differ, depending on the bacterial species: FtsK-mediated activation of Xer recombination in *H. influenzae* obeys the *E. coli* paradigm [21], whereas in *B. subtilis*, the model bacteria for firmicutes (formerly known as low GC-content Gram-positive bacteria), neither of the two FtsK analogs (SpoIIIE and YtpT) appears able to drive Xer recombination [26]. Several attempts have

**Editor:** Dhruba K. Chattoraj, National Cancer Institute, United States of America

**Received** March 16, 2007; **Accepted** June 4, 2007; **Published** July 13, 2007

**Copyright:** © 2007 Le Bourgeois et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

**Abbreviations:** Km<sup>R</sup>, kanamycin resistance; ORF, open reading frame; WT, wild type

<figure><img src="image_87.png" /></figure>

<footnote>* To whom correspondence should be addressed. E-mail: pascal.lebourgeois@ibcg.biotoul.fr</footnote>

<footnote>† Dedicated to my colleague and friend Frédérique Messud (1962–2005)</footnote>
## Author Summary

In bacteria, genetic information is mainly carried by a single circular chromosome. The replication of this circular molecule sometimes leads to the formation of a chromosome dimer unable to segregate in the daughter cells during the division process. In the bacterial model *E. coli*, chromosome dimers are resolved in monomers by site-specific recombination: two recombinases (XerC and XerD) cooperatively catalyze the recombination at a chromosomal site (*dif*), located at the junction of the two replication arms. This recombination is intimately coupled to cell division by the control of the septal protein FtsK. Xer recombination machinery as described in *E. coli* appears highly conserved among bacterial species. We show by comparative genomics and genetic studies that *Streptococci* use an alternative Xer recombination system, renamed XerS/dif<sub>SL</sub>, which is composed of a single recombinase phylogenetically unrelated to XerCD proteins and a noncanonical *dif* site. We also demonstrate that the streptococcal FtsK protein localizes at the division septum and operates the XerS/dif<sub>SL</sub> recombination. This is the first identification of an alternative Xer recombination system in prokaryotes to out knowledge, which might indicate that other unusual chromosome dimer resolution systems could exist in bacterial phyla where a canonical *dif* site is not detected.

been made to identify the Xer recombination machinery in *Streptococci*, a taxonomic group belonging to firmicutes and comprising major pathogens [29] as well as innocuous food-grade species of major industrial importance [30,31]. These studies revealed putative XerCD recombinases but failed to identify a *dif* site [32,33].

We show here, by comparative genomics and functional analyses in *L. lactis*, *S. pneumoniae*, and *E. coli*, that *Streptococci* possess alternative Xer recombination machinery phylogenetically unrelated to the classical XerCD/dif system. This machinery involves a single tyrosine recombinase (XerS) and an atypical *dif* site (*dif*<sub>SL</sub>), both located on a single genetic module. We also show that, in contrast to *B. subtilis*, the streptococcal FtsK protein localizes at the division septum and controls the XerS/dif<sub>SL</sub> recombination.

## Results

### Identification of the Streptococcal *dif* Site by Comparative Genomics

Assuming that Xer recombination is highly conserved in eubacteria with a significant homology of the *dif* sites even between distantly related species [26], we performed an in silico search for putative *dif* sites in several completely sequenced firmicutes genomes. Candidate *dif* sites should: (i) have a significant similarity with that of *B. subtilis* (*dif*<sub>BS</sub>), (ii) occur only once per genome, and (iii) be localized in the replication terminus (*terC*), defined as the chromosomal region located opposite the replication origin (*oriC*) where compositional strand biases switch sign [34,35]. Using these rules, a canonical *dif* site could be identified in all species analyzed except for *Streptococci* and *Lactococci* (Table 1). We therefore used an alternative three-step approach based on comparative genomics to identify the streptococcal/lactococcal *dif* site (Figure 1). The *terC* region for three streptococcal genomes was localized (Figure 1A) using a cumulative GC skew diagram [34], and a comparison of the 10-kb region encompassing the GC skew shift was performed (Figure 1B).

This analysis revealed a 2-kb segment that showed significant similarity within the three species (>70% identity at the DNA level) and included a 356-amino-acid tyrosine-recombinase-encoding gene (annotated *ymfD* on the *L. lactis* IL1403 genome [36] but hereafter renamed *xerS*) preceded by a ~50-bp highly (>90%) conserved sequence. When used to scan 49 genomes of other streptococcal species (Figure 1C), this ~50-bp fragment revealed a 31-bp consensus sequence (hereafter named *dif*<sub>SL</sub>) with weak homology to the *B. subtilis* or *E. coli* *dif* sites, but with an overall structure resembling the DNA targets for tyrosine recombinases (i.e., two imperfect inverted repeats separated by a 6-8-bp central sequence).

Comparative analysis of the genetic context in the 10-kb *terC* region of different streptococcal species revealed notable features strongly suggesting that streptococcal Xer recombination machinery is defined by one genetic module, corresponding to the *dif*<sub>SL</sub> site followed by one of its dedicated recombinases (Figure 2). The physical link between *dif*<sub>SL</sub> and *xerS* open reading frame (ORF) was found to be preserved among all *Streptococci* for which there is sequence data, and no genetic element other than *dif*<sub>SL</sub>-*xerS* was conserved in the 10-kb *terC* region. In addition, the genes surrounding *dif*<sub>SL</sub>-*xerS* did not show a preferred orientation that might indicate possible cotranscription with the *xerS* gene. Moreover, the *xerS* ORF often exhibits a putative ρ-independent transcription terminator at its end. These observations indicate that *xerS* is unlikely to be part of an operon and suggest that the *dif*<sub>SL</sub>-*xerS* pair behaves as an individual genetic module.

### In Vivo Characterization of the *dif*<sub>SL</sub> Site

The candidate *dif*<sub>SL</sub> site was tested for its ability to support site-specific recombination in *L. lactis* and *S. pneumoniae* by intermolecular recombination assays. A 37-bp synthetic sequence encompassing the putative 31-bp lactococcal *dif*<sub>SL</sub> site was cloned in a plasmid that does not replicate in firmicutes (pCL52, Table S1). The resulting construction (pCL235, Table S1) was used to transform a wild-type (WT) strain of *L. lactis* (MG1363, Table S1). In contrast to pCL52, which did not yield transformant, pCL235 produced transformants at an efficiency representing 1% of the efficiency attained with a replicative plasmid (unpublished data). This demonstrates that the putative *dif*<sub>SL</sub> site was capable of rescuing pCL235, presumably by promoting integration of exogenous DNA into the lactococcal chromosome. When transformed into a *recA* strain (VEL1122, Table S1), plasmid pCL235 was also rescued with the same efficiency as in the WT strain (unpublished data), indicating that plasmid integration occurred in a RecA-independent manner. Moreover, as judged by Pulsed-Field gel Electrophoresis analysis (Figure S1, lanes 2 and 4), pCL235 integrated into the chromosome of both strains at the location predicted for the native *dif*<sub>SL</sub> site. Thus, the 37-bp sequence appeared to contain the DNA target of a site-specific recombination system. The *dif*<sub>SL</sub>-mediated site-specific integration was also demonstrated to be a general process in *Streptococci*, since plasmid pGh9, a temperature sensitive replication (*repA*<sup>ts</sup>) mutant containing either the lactococcal 37-bp sequence described above (pCL231, Table S1) or its pneumococcal counterpart (pCL403, Table S1), integrated into the chromosomes of *L. lactis* and *S. pneumoniae* under nonpermissive conditions with comparable efficiencies (respectively 4.88 ×

<figure><img src="image_88.png" /></figure>
<table><caption>**Table 1.** Localization of *dif* Sites in Some Firmicutes</caption><thead><tr><th rowspan="2">Bacterial Species</th><th colspan="3">Sequence</th><th rowspan="2">Site Position<sup>a</sup></th><th rowspan="2">GC Skew Shift Position</th></tr><tr><th>CodV (XerC) Arm</th><th>Core</th><th>RipX (XerD) Arm</th></tr></thead><tbody><tr><td>B. subtilis 168</td><td>acttcctagaa</td><td>tatata</td><td>ttatgtaaact</td><td>1,941,752</td><td>1,941,000</td></tr><tr><td>B. anthracis Ames</td><td>actgcctataa</td><td>tatata</td><td>ttatgttaact</td><td>2,507,756</td><td>2,506,000</td></tr><tr><td>B. anthracis Ames ancestor</td><td>actgcctataa</td><td>tatata</td><td>ttatgttaact</td><td>2,507,880</td><td>2,505,000</td></tr><tr><td>B. anthracis A2012</td><td>actgcctataa</td><td>tatata</td><td>ttatgttaact</td><td>2,981,384</td><td>2,980,000</td></tr><tr><td>B. anthracis Sterne</td><td>actgcctataa</td><td>tatata</td><td>ttatgttaact</td><td>2,507,809</td><td>2,505,000</td></tr><tr><td>B. cereus ATCC 10987</td><td>actgcctataa</td><td>tatata</td><td>ttatgttaact</td><td>2,590,351</td><td>2,590,000</td></tr><tr><td>B. cereus ATCC 14579</td><td>actgcctataa</td><td>tatata</td><td>ttatgttaact</td><td>2,681,362</td><td>2,681,000</td></tr><tr><td>B. halodurans C-125</td><td>ggttcctataa</td><td>tatata</td><td>ttatgtaaact</td><td>2,243,235</td><td>2,242,000</td></tr><tr><td>B. thuringiensis 97-27</td><td>actgcctataa</td><td>tatata</td><td>ttatgttaact</td><td>2,560,283</td><td>2,559,000</td></tr><tr><td>E. faecalis V583</td><td>actttgtataa</td><td>tgtata</td><td>ttatgttaact</td><td>1,550,523</td><td>1,550,000</td></tr><tr><td>L. innocua Clip11262</td><td>acttcctataa</td><td>tatata</td><td>ttatgtaaact</td><td>1,449,102</td><td>1,449,000</td></tr><tr><td>L. monocytogenes EGD-e</td><td>acttcctataa</td><td>tatata</td><td>ttatgtaaact</td><td>1,442,174</td><td>1,443,000</td></tr><tr><td>L. monocytogenes 4b F2365</td><td>acttcctataa</td><td>tatata</td><td>ttatgtaaact</td><td>1,421,892</td><td>1,430,000</td></tr><tr><td>Lb. johnsonii NCC533</td><td>aattcgtataa</td><td>tatata</td><td>ttatgtaaagt</td><td>1,099,730</td><td>1,100,000</td></tr><tr><td>Lb. plantarum WCFS1</td><td>actttgtataa</td><td>tatata</td><td>ttatgtaaact</td><td>1,655,148</td><td>1,650,000</td></tr><tr><td>Oceanobacillus iheyensis HTE831</td><td>acttcctataa</td><td>taataa</td><td>ttatgtctact</td><td>1,772,251</td><td>1,780,000</td></tr><tr><td>S. aureus MW2</td><td>acttcctataa</td><td>taata</td><td>ttatgtaaact</td><td>1,384,891</td><td>1,390,000</td></tr><tr><td>S. aureus N315</td><td>acttcctataa</td><td>tatata</td><td>ttatgtaaact</td><td>1,382,903</td><td>1,390,000</td></tr><tr><td>S. aureus Mu50</td><td>acttcctataa</td><td>tatata</td><td>ttatgtaaact</td><td>1,459,232</td><td>1,470,000</td></tr><tr><td>S. aureus MRSA252</td><td>acttcctataa</td><td>tatata</td><td>ttatgtaaact</td><td>1,447,645</td><td>1,450,000</td></tr><tr><td>S. aureus MSSA476</td><td>acttcctataa</td><td>tatata</td><td>ttatgtaaact</td><td>1,413,445</td><td>1,430,000</td></tr><tr><td>S. epidermitis ATCC 12228</td><td>acttcctataa</td><td>tatata</td><td>ttatgtaaact</td><td>1,070,745</td><td>1,095,000</td></tr><tr><td>L. lactis IL1403</td><td>No homology</td><td></td><td></td><td>NA</td><td>NA</td></tr><tr><td>Streptococcus (nine species)</td><td>No homology</td><td></td><td></td><td>NA</td><td>NA</td></tr></tbody></table>

Variable nucleotides are underlined. Homology search was performed using the *B. subtilis* (*dif*<sub>BS</sub>) site as query against complete genomes available at NCBI (http://www.ncbi.nlm.nih.gov/sutils/genom_tree.cgi?; parameters [with “Megablast” designated as “off” and “Filter” as “none”]).

<sup>a</sup>Indicates first bp of the *dif* site.

NA, not applicable.

doi:10.1371/journal.pgen.0030117.t001

$10^{-2}$ [± 2.33 × $10^{-2}$] cell⁻¹ and 2.67 × $10^{-2}$ [± 1.55 × $10^{-2}$] cell⁻¹). However, it should be mentioned that location of the insertion site of pCL403 has not been verified in *S. pneumoniae*.

The minimal size of the *dif*<sub>SL</sub> site was determined in *L. lactis* by scoring the integration efficiency of pGh9 containing variants of the *dif*<sub>SL</sub> site (Table 2). Reducing the length of the *dif*<sub>SL</sub> region from 48 to 31 bp did not alter the plasmid integration efficiency, indicating that the strongly conserved T located 13 bp away from the 31-bp consensus sequence (Figure 1C) was not part of the *dif*<sub>SL</sub> site. However, removing the two external bp from both sides of the 31-bp consensus sequence (Table 2, *dif*-8) led to a 100-fold decrease in integration efficiency, though this sequence was still proficient in site-specific recombination at the native *dif*<sub>SL</sub> site (Figure S1, lanes 3 and 5). Finally, deleting two nucleotides at either side of the 31-bp consensus sequence led to a 2-fold (Table 2, compare *dif*-7 to *dif*-5; Wilcoxon test, *p* < 0.003) or 4-fold (Table 2, compare *dif*-6 to *dif*-5; Wilcoxon test, *p* < 0.01) reduction in integration frequency. Together, these results led us to propose that the 31-bp consensus sequence defines the authentic *dif*<sub>SL</sub> site.

### Recombination at *dif*<sub>SL</sub> Requires One Recombinase, XerS

Given that predictive analyses revealed XerS as the prime candidate for the actual Xer recombinase, a recombination assay was performed in *S. pneumoniae* to test if XerS was needed for recombination at *dif*<sub>SL</sub>. This streptococcal species was selected mainly for its facility to construct mutants

compared to *L. lactis*. The recombination at the *dif*<sub>SL</sub> site was totally abolished in a *xerS* mutant (strain S501, Table S1; Materials and Methods), with undetectable integration of pCL403, demonstrating that XerS was one catalytic recombinase of the XerS/*dif*<sub>SL</sub> system. To test whether XerS was the only recombinase involved in the Xer catalytic machinery, XerS/*dif*<sub>SL</sub> recombination was assayed in *E. coli* using an excision assay previously developed for the genetic analysis of the *E. coli* *dif* site activity [37]. Briefly, the native *E. coli* *dif* site was replaced by a cassette containing two directly repeated lactococcal *dif*<sub>SL</sub> sites flanking a kanamycin resistance (Km<sup>R</sup>) gene (strain E359, Table S1), and the excision frequency (cell⁻¹ generation⁻¹; Materials and Methods) was determined by counting the number of Km<sup>S</sup> recombinants at different generations during serial cultures (Figure 3A). In absence of the lactococcal XerS recombinase, almost no recombination was observed (<0.006% cell⁻¹ generation⁻¹) indicating that XerCD of *E. coli* do not recombine *dif*<sub>SL</sub>. In contrast, introduction of a plasmid expressing the lactococcal *xerS* gene (pCL297, Table S1) increased the excision frequency to 10% cell⁻¹ generation⁻¹ (Figure 3A), a value close to the excision frequency observed in *E. coli* when using the native XerCD/*dif* system [3]. In addition, the excision frequency was not significantly altered in *E. coli* *xerC* or *xerD* mutant (Figure 3A). This indicates that fortuitous interaction between XerS and *E. coli* XerC or XerD recombinases is unlikely to account for recombination at *dif*<sub>SL</sub> sites. However, as recombination assay has not been performed on a *xerC* *xerD* double mutant, this cannot be totally ruled out. XerS also promoted

<figure><img src="image_89.png" /></figure>
<figure><img src="image_90.png" /></figure>

<table><thead><tr><th>Gene</th><th>Transposon</th><th>Transposon Type</th><th>Genome</th><th>Transposon Site</th><th>Transposon Type</th><th>Genome</th><th>Transposon Site</th></tr></thead><tbody><tr><td>TTgAAAAACTCCTATAAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATATACAAgTCT</td><td>ATgCT</td><td>S. pneumoniae R6, D39, and TIGR4</td><td></td></tr><tr><td>TTgAAAAACTCCTATAAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATATACAAgTCT</td><td>ATgCT</td><td>S. pneumoniae 670-6B *</td><td></td></tr><tr><td>TTgAAAAACTCCTATAAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATATACAAgTCT</td><td>ATgCT</td><td>S. pneumoniae 23F *</td><td></td></tr><tr><td>TTAAAAAAACTCTATgAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATAAATAAgTCT</td><td>ATgCT</td><td>S. mitis NCTC12261 *</td><td></td></tr><tr><td>TTTCgATTACTCTCTATC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATATATCTAgAT</td><td>ATgCT</td><td>S. sanguinis SK36</td><td></td></tr><tr><td>CTATATATgCCCTCTCTC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATATATCTAgAT</td><td>ATgCT</td><td>S. gordonii Challis NCTC7868 *</td><td></td></tr><tr><td>AgAAgAACACCTCATAAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>gAAATATAAggg</td><td>TgTgAT</td><td>S. thermophilus CNRZ1066, LMD-9, and LMG18311</td><td></td></tr><tr><td>TCTGATATCCCCACAAAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>ACTTATgTAAgAT</td><td>ATTgCT</td><td>S. suis 89/1591 *</td><td></td></tr><tr><td>TCTAATATCCCCACAAAC</td><td>ATTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>CTTATATAAggt</td><td>TTATgCT</td><td>S. suis P1/17 *</td><td></td></tr><tr><td>gAAATAAAAggTTATCg</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>ATAATgTgTAAg</td><td>TgTgCT</td><td>S. mutans UA159</td><td></td></tr><tr><td>TCgAgTATTTgTTATAACT</td><td>CTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATATATAggg</td><td>CgTgAT</td><td>S. sobrinus 6715 *</td><td></td></tr><tr><td>TCTAATATTTCCCATAAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>ACATTCAAgg</td><td>ATggtT</td><td>S. uberis 0140J *</td><td></td></tr><tr><td>ACTATTAAATAATTTTAT</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATACCCCAAg</td><td>TgTgAT</td><td>S. equi subsp equi *</td><td></td></tr><tr><td>gCAAAATAACTATCACAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATATCTAgTT</td><td>TAAAAA</td><td>S. equi subsp. zooepidemicus *</td><td></td></tr><tr><td>AgAgATCTgTCTCgTAAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATATTTATAgg</td><td>TgTgAT</td><td>S. pyogenes M1</td><td></td></tr><tr><td>AgAgATTgTCTCgTgAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATATTTATAgg</td><td>TgTgAT</td><td>S. pyogenes M3</td><td></td></tr><tr><td>AgAgATCTgTCTCgTAAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATATTTATAgg</td><td>TgTgAT</td><td>S. pyogenes M18 (strain M8232)</td><td></td></tr><tr><td>AgAgATCTgTCTgTgAAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>AATATTTATAgg</td><td>TgTgAT</td><td>S. pyogenes M5 Manfredo *</td><td></td></tr><tr><td>TCCTgTTTTTAACTTTA</td><td>ACTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>gTTATAACAAgg</td><td>TATgAT</td><td>S. agalactiae NEM316 and 515</td><td></td></tr><tr><td>TCCTgTTTTTAACTTTA</td><td>ACTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAAA</td><td>gTTATgATAgAg</td><td>TATgAT</td><td>S. agalactiae 2603V/R</td><td></td></tr><tr><td>CTATAgTTTTAAgTAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAA</td><td>CAATTgg</td><td>ATATCT</td><td>L. lactis subsp lactis IL1403</td><td></td></tr><tr><td>CTATAgTTTTAAgTAC</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAA</td><td>CAATTg</td><td>ATTATCT</td><td>L. lactis subsp cremoris MG1363</td><td></td></tr><tr><td>CTATAAgTTTTAATTACA</td><td>ATCTTTTCg</td><td>AAAAACT</td><td>TAATTTTCTTgAA</td><td>CAATTgg</td><td>ATATCT</td><td>L. lactis subsp cremoris SK11</td><td></td></tr></tbody></table>

<figure><img src="image_91.png" /></figure>

dif (E. coli K12)

dif<sub>SL</sub>

dif (B. subtilis 168]

<caption>**Figure 1.** Identification of the Streptococcal *dif*<sub>SL</sub> Site by Comparative Genomics</caption>

(A) Cumulative GC skew diagrams of three streptococcal genomes. Lla, *L. lactis* IL1403; Spyo, *S. pyogenes* M1 GAS; Spn, *S. pneumoniae* R6.

(B) Multiple DNA comparison is presented for the 10-kb regions encompassing the region of GC skew shift. The result is scaled to the gene organization of the *L. lactis* IL1403 *terC* region, and the conserved ORF is indicated in black. The lactococcal ORFs A, B, and C, not found in other streptococcal genomes, showed significant homology to the YmfD protein when fused together, suggesting ancient duplication of the *ymfD* region in *L. lactis*. The conserved ~50-bp sequence is indicated by an asterisk.

(C) Sequence similarity of putative *dif*<sub>SL</sub> sites from 49 publicly available streptococcal genomes (December 2006) and comparison with the *E. coli* and *B. subtilis* *dif* sites are presented. Nucleotides conserved in all except one species are indicated in bold, and those conserved in all species are indicated in bold and underlined. *S. pyogenes* M1 strains were M1 GAS, MGAS10750, MGAS5005, and MGAS10394. *S. pyogenes* M3 strains were MGAS315, MGAS2096, MGAS10270, MGAS9429, MGAS6180, M49–591, and SSI-1. *S. agalactiae* sequences of strains A909, H36B, COH1, CJB111, and 18RS21 were identical to strain 2603V/R. Asterisks indicate unfinished genomes.

doi:10.1371/journal.pgen.0030117.g001

intermolecular recombination between one lactococcal *dif*<sub>SL</sub> site replacing the native *E. coli* *dif* site on the chromosome (strain E368, Table S1) and a second *dif*<sub>SL</sub> site located on a nonreplicative plasmid (unpublished data). Together, these data demonstrated that XerS is the only streptococcal tyrosine recombinase required to catalyze intra- and intermolecular recombination between *dif*<sub>SL</sub> sites.

A phylogenetic analysis of all tyrosine recombinases present in the sequenced genome of five streptococcal species revealed another integrase conserved among *Streptococci*. This atypical recombinase, more related to phages' integrases (Figure S2) and previously identified as XerD in *S. pneumoniae* [33], lacks the extreme C-terminal region and two amino acids of the catalytic tetrad R-H-R-Y [38]. When tested alone

<figure><img src="image_92.png" /></figure>
<figure><img src="image_93.png" /><figcaption>**Figure 2.** Gene Context Analysis of the 10-kb *terC* Region of Different Streptococcal Species</figcaption></figure>

Lla, *L. lactis* IL1403; Spn, *S. pneumoniae* R6; Spy, *S. pyogenes* M1 GAS; Sagal, *S. agalactiae* NEM316; Sth, *S. salivarius* subsp. *thermophilus* CNRZ1066; Smut, *S. mutans* UA159. The ORF coding for the putative tyrosine recombinase is shown in black. Only putative p-independent transcription terminators with free energy (ΔG) < -12 kcal.mol⁻¹ are indicated.

doi:10.1371/journal.pgen.0030117.g002

in *E. coli*, YnbA (the lactococcal ortholog of *S. pneumoniae* XerD) showed no intra- or intermolecular recombination activity on *dif*<sub>SL</sub> and did not influence the recombination process when coexpressed with XerS. Moreover, it did not bind specifically to the lactococcal *dif*<sub>SL</sub> site in vitro (unpublished data). Therefore, YnbA is unlikely to belong to the streptococcal Xer system.

### XerS/dif<sub>SL</sub> and Chromosome Dimer Resolution in *E. coli*

Although the XerS/dif<sub>SL</sub> system involves only one recombination, as do the Cre/loxP and Flp/FRT systems, its location at the *terC* region of streptococcal chromosomes strongly suggests that it functions to resolve chromosome dimers. To examine whether XerS/dif<sub>SL</sub> can substitute the XerCD/dif system in *E. coli*, we used the growth competition assays (Figure 3B) previously developed to show that XerCD/dif resolved chromosome dimers in *E. coli* [3,39]. For that purpose, we constructed *E. coli* strains containing or not one lactococcal *dif*<sub>SL</sub> site replacing the native *dif* site. The

strain harboring a complete streptococcal Xer system (E368, Table S1) showed a growth advantage of 10% generation⁻¹ when competed with its isogenic strain missing the *dif*<sub>SL</sub> site (E367, Table S1). As found for XerCD in *E. coli* [37], this selective benefit matches the excision frequency of the Km<sup>R</sup> cassette in the excision assay described above. In addition, strain E368 showed no growth defect compared to a strain harboring a functional XerCD/dif system (E375, Table S1). These results were correlated with cell morphology changes: strain E367 retained the filamentation phenotype of an *E. coli* strain defective in Xer recombination, while the strain harboring the complete XerS/dif<sub>SL</sub> system displayed a WT cell morphology (unpublished data). Thus, the XerS/dif<sub>SL</sub> system can substitute XerCD/dif in *E. coli* to resolve chromosome dimers.

Chromosome dimers in *E. coli* are mostly formed by homologous recombination [2]. As a *recA* mutation also drastically reduces the XerCD-mediated recombination at *dif* [2,37], this argued toward the fact that chromosome dimer is mandatory for creating the conditions necessary for a recombination between two directly repeated *dif* sites. Such dependence was investigated for the XerS system using the same *E. coli* excision assay [37]. The frequency of the Km<sup>R</sup> cassette excision by the XerS system fell from 10% to less than 0.6% cell⁻¹ generation⁻¹ (Figure 3A) in a *recA* derivative of the E359 strain (strain E379, Table S1). As this 20-fold reduction of recombination efficiency was similar to that observed in *E. coli* [37], this strongly suggests that, as for the XerCD/dif system, XerS can perform efficient recombination only when *dif*<sub>SL</sub> sites are located on chromosome dimers.

### XerS/dif<sub>SL</sub> Recombination Depends on the Septal Protein FtsK

All streptococcal genomes sequenced so far contain one ORF encoding a protein homologous to the 787-amino-acid *B. subtilis* protein SpoIIIE. These SpoIIIE-like proteins (hereafter named FtsK<sub>SL</sub>) range from 758 (*S. mutans*) to 816 (*S. agalactiae*) amino acids in length and retain the structural signatures of proteins from the FtsK-HerA superfamily [40]: they contain a ~180-amino-acid N-terminal region of weak similarity that includes four predicted transmembrane domains and a strongly conserved ~500-amino-acid C-terminal region corresponding to the DNA translocase domain (unpublished data). The cellular localization of FtsK<sub>SL</sub> was determined in *L. lactis* using GFP fusions,

<table><caption>**Table 2.** Chromosomal Integration Frequencies of *dif*<sub>SL</sub> Variants in *L. lactis*</caption><thead><tr><th>Name (Plasmid<sup>a</sup>)</th><th>Size (bp)</th><th>Sequence<sup>b</sup></th><th>Integration Frequency (Integration Frequency per Cell)<sup>c</sup></th></tr></thead><tbody><tr><td>dif-1 (pCL247)</td><td>48</td><td>TAACATCTTTCCgAAAAACTgTAATTTTCTTGACAATTggATATCTCT</td><td>2.86 &times; 10<sup>-2</sup> (&plusmn; 7.61 &times; 10<sup>-3</sup>)<sup>d</sup></td></tr><tr><td>dif-5 (pCL251)</td><td>31</td><td>ATCTTTCCgAAAAACTgTAATTTTCTTGACA</td><td>2.67 &times; 10<sup>-2</sup> (&plusmn; 1.03 &times; 10<sup>-2</sup>)</td></tr><tr><td>dif-6 (pCL279)</td><td>29</td><td>CTTTCCgAAAAACTgTAATTTTCTTGACA</td><td>5.12 &times; 10<sup>-3</sup> (&plusmn; 2.58 &times; 10<sup>-3</sup>)</td></tr><tr><td>dif-7 (pCL281)</td><td>28</td><td>ATCTTTCCgAAAAACTgTAATTTTCTTGAA</td><td>9.28 &times; 10<sup>-3</sup> (&plusmn; 2.98 &times; 10<sup>-3</sup>)</td></tr><tr><td>dif-8 (pCL233)</td><td>27</td><td>CTTTCCgAAAAACTgTAATTTTCTTGAA</td><td>2.34 &times; 10<sup>-4</sup> (&plusmn; 1.45 &times; 10<sup>-4</sup>)</td></tr></tbody></table>

<figure><img src="image_94.png" /></figure>

<footnote><sup>a</sup>See Materials and Methods for detailed construction of the plasmids.</footnote>

<footnote><sup>b</sup>Conserved nucleotides among *Streptococci* are indicated as in Figure 1C.</footnote>

<footnote><sup>c</sup>See Materials and Methods. Plasmid pGh9 integration frequency was 2.61 &times; 10<sup>-5</sup> (9.53 &times; 10<sup>-6</sup>).</footnote>

<footnote><sup>d</sup>Standard deviation (σ<sup>n-1</sup>).</footnote>

<footnote>doi:10.1371/journal.pgen.0030117.t002</footnote>
<figure><img src="image_95.png" /></figure>

<figure><img src="image_96.png" /><figcaption>**Figure 3.** XerS/dif<sub>SL</sub> Recombination in *E. coli*</figcaption></figure>

(A) Km<sup>R</sup> cassette excision mediated by XerS in *E. coli* in different genetic backgrounds is shown. The +/− signs indicated presence/absence of plasmid expressing the *L. lactis* xerS (pCL297) or the *E. coli* ftsK (pCL263) genes. When available the number of independent experiments (n) is indicated below each excision frequency mean value. Error bars correspond to the standard deviation (σ<sup>n-1</sup>).

(B) Effect of the lactococcal XerS/dif<sub>SL</sub> system on chromosome dimer resolution in *E. coli* as measured by growth competition assays is presented. E368 (XerS<sup>+</sup> *dif*<sub>SL</sub><sup>+</sup>) was mixed with E367 (XerS<sup>+</sup> *dif*<sub>SL</sub><sup>−</sup>, squares), E378 (XerS<sup>+</sup> *dif*<sub>SL</sub><sup>+</sup> *ftsK<sub>C</sub><sup>−</sup>, triangles), or E375 (WT strain with XerS, circles) at a ratio of 1:1 and grown in serial culture for 60 generations. Values were calculated from two independent assays.
doi:10.1371/journal.pgen.0030117.g003

corresponding to the GFP protein fused to the C-terminal of the full-length (FtsK<sub>1-763</sub> - GFP) or N-terminal region (FtsK<sub>1-181</sub> - GFP) of lactococcal FtsK<sub>SL</sub>. Both GFP fusions clearly localized at the septum of *L. lactis* (Figure 4), indicating that as expected, FtsK<sub>SL</sub> localizes at the lactococcal division septum

<figure><img src="image_97.png" /><figcaption>**Figure 4.** Subcellular Localization of FtsK<sub>SL</sub>-GFP Proteins in *L. lactis*</figcaption></figure>

Phase-contrast (A) and fluorescence (B) microscopy of *L. lactis* NZ900 overexpressing full-length lactococcal FtsK-GFP (upper) or N-ter Ftsk-GFP (lower) are presented. Cells were analyzed by microscopy on mid-exponential growth phase.
doi:10.1371/journal.pgen.0030117.g004

and its 181 amino acids containing the four transmembrane domains were sufficient to drive this localization.

The control of XerS-mediated recombination by FtsK was examined in *S. pneumoniae* and *E. coli*. For that purpose, strains expressing FtsK proteins deleted of their C-terminal part were constructed (*ftsK*<sub>C</sub> mutants, only the first 405 amino acids and the first 316 amino acids of FtsK are synthesized in *S. pneumoniae* and *E. coli* respectively; Materials and Methods). Surprisingly, during the construction of the *ftsKC* mutants in *S. pneumoniae*, insertions of the Mariner minitransposon were obtained into the C-terminal or the N-terminal domain of FtsK<sub>SL</sub>. This suggests that neither the C- nor the N terminus is essential for the growth of this bacterium, though all pneumococcal *ftsK* mutants were severely impaired in growth rate and cell viability as *xerS* mutants (unpublished data). In *S. pneumoniae*, XerS/dif<sub>SL</sub> recombination depended on the C-terminal part of FtsK<sub>SL</sub>, because integration of the *repA*<sup>ts</sup> plasmid containing the pneumococcal *dif*<sub>SL</sub> site (pCL403), though not totally abolished as for the *xerS* mutant, became severely impaired in the *ftsK*<sub>C</sub> mutant (S502, Table S1), with an efficiency of 1.52 × 10<sup>-4</sup> (± 3.5 × 10<sup>-4</sup>) cell<sup>-1</sup> corresponding to less than 1% of the integration efficiency of the WT strain. Similar observations were made in the *ftsK*<sub>C</sub> mutant of

<figure><img src="image_98.png" /></figure>
*E. coli* (E372, Table S1), using the excision assay. The excision frequency of the *difSL*-Km<sup>R</sup>- *difSL* cassette dropped from 10% in WT strain to 0.1% cell<sup>-1</sup> generation<sup>-1</sup> in the *ftsK<sub>C</sub>* strain (Figure 3A). This decrease was unambiguously associated to the lack of the C-terminal part of FtsK, since expression of the full-length *E. coli* *ftsK* gene carried on a pBAD-derivative plasmid (pCL263, Table S1) restored Km<sup>R</sup> cassette excision to nearly the WT frequency (Figure 3A). In addition, results from growth competition assays (Figure 3B) or cell morphology observations (unpublished data) also showed that the XerS system was unable to resolve chromosome dimers in *E. coli* in absence of the C-terminal domain of FtsK. Together our data demonstrated that XerS/difSL recombination in *S. pneumoniae* and *E. coli*, as well as dimer resolution in *E. coli*, depends on FtsK.

Discussion

In this work, we provide experimental evidence that *Streptococci* possess an unconventional Xer recombination machinery that requires only one tyrosine recombinase, XerS, to catalyze the site-specific recombination at a 31-bp sequence *dif*<sub>SL</sub>. This raises an important question as to whether this system is orthologous to the “classical” *E. coli* XerCD system found in most bacterial species, including many other firmicutes. Not only does XerS differ significantly in length and primary sequence from members of the XerCD recombinases family (unpublished data), but *dif*<sub>SL</sub> also differs in length and shows a weak similarity with the *E. coli* or *B. subtilis* *dif* sites (Figure 1C). Moreover, *dif*<sub>SL</sub> is located immediately upstream the *xerS* coding sequence in all streptococcal and lactococcal species analyzed. Such genetic organization contrasts with that of classical XerCD systems, with the two recombinases encoded by genes located far from each other and distant from *dif*, and is more comparable to the integration modules of mobile elements such as integrons [41], bacteriophages such as P1 [42] or mycobacteriophage L5 [43], and some ICEs such as the *clc* element from *Pseudomonas* [44] or CTnDOT from *Bacteroides* [45]. As *Streptococci* and *Lactococci* (together defining the taxonomic family of *Streptococcaceae*) represent a rather homogeneous phylogenetic group among firmicutes when compared to other genera such as *Clostridium* or *Lactobacillus* [46,47], we speculate that acquisition of the XerS system might have replaced the “classical” XerCD system at the time of or soon after their split from other firmicutes, with this event representing one landmark of this separation.

As demonstrated in this study, the cis-organization of the *difSL*-xerS module is not mandatory for efficient recombination, but this probably reveals a selective pressure to maintain that arrangement. Although at present the xerS transcription start point location is unknown, we speculate that difSL either lies between the xerS ORF and its promoter or is part of the xerS promoter. If this is true, this unusual arrangement might reflect a regulatory mechanism in which binding of XerS to difSL might autoregulate xerS expression. Alternatively, as it has been recently observed that some filamentous phages [48] or genetic islands [49] can divert the XerCD recombination system to integrate themselves at the chromosomal dif site of several bacteria, another hypothesis could be that the difSL-xerS arrangement might serve to prevent insertion of additional genetic material at difSL, because such event would

separate the *xerS* ORF from its promoter and lead to inactivation of the chromosome dimer resolution system.

With only one catalytic recombinase involved in the recombination reaction, the XerS system is more similar to Cre/loxP and Flp/FRT than to XerCD/dif. However, XerS retains particular features that could indicate alternative mechanism in the recombination process. For instance, in vivo characterization of the *difSL* site in *L. lactis* revealed an asymmetry in its organization, with left and right arms differing in length (the left arm being 2-bp longer than the right one) as well as in nucleotide sequence (Table 2). This differs from loxP and FRT sites, which contain two perfectly symmetrical 13-bp arms surrounding the core region [50,51]. How a single recombinase can accommodate dissimilar binding sites to perform the DNA strand exchange reaction without accessory factor has to be analyzed, but we speculate that asymmetry of the *difSL* site might have a role in the control of this strand exchange order.

Though we did not provide direct experimental data demonstrating that XerS/difSL is involved in chromosome dimer resolution in *Streptococci*, several lines of evidence strongly suggest that dimer resolution is the primary task of this site-specific recombination system. First, classical XerCD recombinases and canonical *dif* site are not present in streptococcal/lactococcal genomes but substituted by the XerS/difSL recombination module at the chromosomal location predicted for a site-specific recombination system acting on chromosome dimer resolution. Second, to catalyze the strand exchange reaction XerS seems to require at least one of the two difSL sites located on the chromosome, because recombination between two difSL sites contained within a multicopy plasmid with theta replication (pSC101 derivative in *E. coli*, and pAMB1 in *L. lactis*, unpublished data) could be detected neither in *E. coli* nor in *L. lactis*. At last, not only is XerS/difSL able to resolve chromosome dimers in *E. coli* as efficiently as the native XerCD/dif system (Figure 3B), but XerS/difSL recombination also hinged on formation of chromosome dimers, as revealed by the RecA-dependency of the Km<sup>R</sup> cassette excision (Figure 3A), with the excision efficiency exactly matching the frequency of chromosome dimers resolution.

We also demonstrated that, in contrast to SpoIIIE from *B. subtilis* that only infrequently (~6%) concentrates at the vegetative septum [52] and is not involved in the Xer recombination [26], the streptococcal FtsK<sub>SL</sub> protein localizes at the division septum and still directs the XerS/difSL recombination, as dimer resolution and intra- or intermolecular recombination were drastically inhibited in *E. coli* and *S. pneumoniae* cells lacking the C-terminal part of FtsK. Although our preliminary analyses of the pneumococcal *ftsK* mutants need to be confirmed, the ability to obtain viable cells depleted of FtsK suggests that neither the N- nor the C terminus of the protein is essential in *Streptococci*. As essentiality of FtsK seems to be species dependent, with only the N-terminal part in *E. coli* and the C terminus in *C. crescentus* being essential [53,54], we hypothesize that activity of FtsK, though still involved in cell division and DNA translocation, could slightly differ among the different bacteria. However, it appears that no correlation can be done between essentiality and localization of FtsK to the division septum. In *E. coli* and some other γ-proteobacteria, the C-terminal part of FtsK drives the XerCD recombination

<figure><img src="image_99.png" /></figure>
reaction in two ways: by participating to the formation of the recombination synapse through its DNA translocase activity [14–16] and activating the recombination reaction by direct interaction with XerD [8]. Some of our data strongly indicated that such interaction between FtsK<sub>SL</sub> and XerS is unnecessary to activate the XerS/dif<sub>SL</sub> recombination in *Streptococci*, though this cannot be totally ruled out. First, the XerS-mediated intramolecular recombination frequency at dif<sub>SL</sub> in *E. coli* (Figure 3A) was close to that measured with XerCD/dif [3], suggesting no species specificity for FtsK requirement. This observation contrasts to that made in *E. coli* where the *H. influenzae* FtsK was inefficient in activation of the *E. coli* XerD and vice versa, implying that the FtsK-XerD interaction is highly species specific in these bacteria [21]. In addition, both pneumococcal and lactococcal XerS protein sequences do not contain the amino acids motif (RQ-QQ) found in *E. coli* XerD and involved in its specific interaction between with FtsK [20]. At last, the cassette excision by recombination at dif<sub>SL</sub> in *E. coli*, as well as plasmid integration in *S. pneumoniae*, was not totally abolished in ftsK<sub>C</sub> mutants but dropped to 1% of the recombination activity of WT strains, suggesting that some productive recombination synapses might form independently of FtsK, most probably by the random collision of two *dif* sites. This observation also contrasts the results obtained with the cassette excision assay performed with the *E. coli* XerCD/dif system, wherein no recombination was detected in an ftsK<sub>C</sub> mutant [55], suggestive of the FtsK-mediated activation of the recombination. Our data are more easily accommodated to a model where XerS is unable to form a productive synapse and requires the DNA translocase activity of FtsK<sub>SL</sub> to bring the two dif<sub>SL</sub> sites of a chromosome dimer close to each other and in an active geometry before performing the strand exchange. However, the recombination would not need direct activation by protein interaction between FtsK<sub>SL</sub> and XerS. However, as for the XerCD model [11], our model cannot provide satisfactory explanation to how FtsK is involved in the intermolecular recombination between one dif<sub>SL</sub> site located on a suicide (or *repA*<sup>ts</sup>) plasmid and the chromosomal dif<sub>SL</sub> site, and the mechanism of the FtsK<sub>SL</sub>-mediated control has to be analyzed further.

In conclusion, the discovery of a Xer recombination system phylogenetically unrelated to the classical XerCD system reinforces the idea that chromosome dimer resolution can be viewed as a housekeeping function conserved among bacteria with circular chromosome(s), but that some species can use functional analogs to perform this task. We expect that other bacterial species among those whose genome(s) are missing a canonical *dif* site also contain alternative chromosome dimer resolution systems. Finally, we note that the particularity of the XerS system makes it a valuable candidate for the development of new antibacterial drugs specifically directed against the pathogenic *Streptococci*.

## Materials and Methods

**Plasmids, bacterial strains, and growth conditions.** The plasmids and bacterial strains used in this study are listed in Table S1. *E. coli* strains and plasmids containing synthetic lactococcal or pneumococcal variants of *dif*<sub>SL</sub> sites were constructed using the procedure provided in Text S1. *E. coli* strains were grown at 30 °C in LB medium. Antibiotics were used at the following concentrations: erythromycin (Em) 150 µg ml<sup>-1</sup>, chloramphenicol (Cm) 20 µg ml<sup>-1</sup>, spectinomycin (Spc) 100 µg ml<sup>-1</sup>, kanamycin (Km) 50 µg ml<sup>-1</sup>, and ampicillin (Ap) 25

µg ml<sup>-1</sup>. *L. lactis* strains were grown semi-anaerobically at 30 °C in M17 broth (Merck KGaA, http://www.merck.de) supplemented with 0.5% (w/v) glucose (GM17) and transformed as previously described [56]. Antibiotics used for selection of lactococcal transformants were: Em 1 µg ml<sup>-1</sup>, Cm 5 µg ml<sup>-1</sup>, and Spc 200 µg ml<sup>-1</sup>. *S. pneumoniae* strains were grown in Todd-Hewitt broth (Difco/BD Biosciences, http://www.bdbiosciences.com) supplemented with 0.5% yeast extract (THY) and transformed using synthetic competence-stimulating peptide (CSP) as described [57]. Antibiotic concentrations used for selection of pneumococcal transformants were: Em 0.2 µg ml<sup>-1</sup> and Km 500 µg ml<sup>-1</sup>.

**DNA manipulation.** Restriction and modification enzymes were purchased from New England Biolabs (http://www.neb.com) and used as recommended by the supplier. Plasmid DNA from *E. coli* was isolated with the Qiaprep spin kit according to the manufacturer's instructions (Qiagen, http://www.qiagen.com). Chromosomal DNA from *E. coli*, *L. lactis*, and *S. pneumoniae* was isolated with the DNeasy tissue kit according to the manufacturer's instructions (Qiagen). Preparation of lactococcal genomic DNA embedded in agarose matrix, Pulsed-Field gel Electrophoresis, and Southern blot with dried agarose gels were performed as previously described [58]. Hybridization signals were detected with a Bioimaging BAS1000 analyzer system (FUJI Photo Film, http://www.fujifilm.com) and analyzed with TINA version 2.07c software (Raytest Isotopenßgeräte GmbH, http://www.raytest.de).

**Genome sequences analyses.** Nucleotide sequences were obtained from NCBI (http://www.ncbi.nlm.nih.gov/genomes/static/eub__g.html), JGI (http://genome.jgi-psf.org/tre_home.html), and the Sanger Institute (http://www.sanger.ac.uk/Projects/Microbes). Cumulative GC skews were obtained from an in-house build program (Laurent Lestrade, Laboratoire de Biologie Moléculaire des Eucaryotes, Toulouse, France). Multiple DNA comparison was performed using MultiPipMaker program [59].

**Intermolecular recombination assays in *L. lactis* and *S. pneumoniae*.** Chromosomal integration assays of *repA*<sup>ts</sup> plasmid pGh9 or *dif*<sub>SL</sub>-containing derivatives in *L. lactis* were performed according to [60]. For *S. pneumoniae*, frozen strains containing the pGh9 or derivatives were grown at 39 °C (water bath) to an OD<sub>600</sub> = 0.3 in THY without antibiotics. Appropriate dilutions were plated on 10 ml of D medium [57] containing 2% of defibrinated horse blood (bioMérieux, http://www.biomerieux.com) and supplemented when appropriate with Em, and plates were incubated at 39 °C. Integration of the pGh9 plasmid was undetectable in *S. pneumoniae* (no colonies were observed when plating 0.1 ml of the undiluted bacterial culture). The integration frequency per cell (ipc) was calculated as the geometric mean of the ratio of colonies on selective versus nonselective plates obtained from five to 19 independent experiments.

**In vitro mariner mutagenesis in *S. pneumoniae*.** Mutagenesis was carried out as described [61]. The target DNA for in vitro transposition of the Km<sup>R</sup> mariner minitransposon pR410 [61] were obtained by PCR reactions using R800 chromosomal DNA as template. The sizes of PCR fragments were: 2.012 bp for *xerS* gene (primers: forward, 5'-TAgAAAACCgATTCTCAgAAATgAgATC-3'; reverse, 5'-gAAgAAgAATTggCCgA AATCAA-3') and 4.053 bp for ftsK<sub>SL</sub> gene (primers: forward, 5'-AAAACAAAgCCTTggTggTgC CT-3'; reverse, 5'-CTTGcgACAAgAAAgggAAA-TTT-3'). The mutagenized PCR fragments were then transformed into strain R800. For each mutagenesis, ten Km<sup>R</sup> transformants were checked by PCR and shown to carry a *mariner* insertion. The accurate insertion position of the transposon, as well as its orientation, was determined by PCR and DNA sequencing as described [61]. The resulting chromosome structures of the selected mutants were: R800 *xerS*, insertion of *mariner* 167 bp downstream the ATG (insertion allows the synthesis of only the first 55 amino acids of XerS); R800 ftsK<sub>C</sub>, insertion of *mariner* 1217 bp downstream the ATG (insertion allows the synthesis of only the first 405 amino acids of FtsK).

**dif**<sub>SL</sub>-Km<sup>R</sup>-dif<sub>SL</sub> **cassette excision in E. coli.** XerS-mediated excision of the *dif*<sub>SL</sub>-Km<sup>R</sup>-dif<sub>SL</sub> was performed according to [37]. Briefly, *E. coli* strains transformed with the XerS expression plasmid pCL297 (or cotransformed with pCL297 and pCL263) were growth in serial cultures at 30 °C (because of the thermosensitive replication of plasmid pCL297) for 60 generations in nonselective LB medium. The ratio of Km<sup>R</sup>/total colonies was measured each 20 generations, and excision frequencies (cell<sup>-1</sup> generation<sup>-1</sup>) deduced according to Perals et al. [37]. For low excision frequencies (<1% cell<sup>-1</sup> generation<sup>-1</sup>), such as those obtained in the absence of XerS in *recA* or ftsK<sub>C</sub> single mutants and *recA* ftsK<sub>C</sub> double mutants, 200 colonies picked from nonselective plates after 85 generations of serial growth were replica plated on Km-containing LB plates, the ratio of Km<sup>R</sup>/

<figure><img src="image_100.png" /></figure>
total colonies was measured, and used to estimate the excision frequencies per generation.

**Growth competition assays in *E. coli*.** Coculture experiments were performed as described by Perals et al. [3], except that the growth temperature was 30 °C (because of the thermosensitive replication of plasmid pCL297). The growth advantage per generation, corresponding to the frequency of chromosome dimer resolution, was calculated from the slope of each competition curve as according to [3]. The strains containing one *dif*<sub>SL</sub> site located at the native position of the *E. coli* *dif* site correspond to Km<sup>S</sup> strains obtained from replica plating from nonselective plates in the *dif*<sub>SL</sub>-Km<sup>R</sup>*dif*<sub>SL</sub> excision experiments described above.

**Subcellular localization of FtsK-GFP fusions in *L. lactis*.** The lactococcal *ftsK* gene was amplified by PCR from chromosomal DNA of *L. lactis* MG1363. The 557-bp PCR fragment (encoding the first 181 amino acids of FtsK; primers: forward, 5'-CATgCCATgggT-gAAAgTAAAAAAATgCCT-3'; reverse 5'-CCATgATTTTAgg-gAAAAgTCC TggAA-3') and the 2303-bp PCR fragment (full-length *ftsK* gene; primers: forward, 5'-CATgCCATgggTgAAAg-TAAAAAAATgCCT-3'; reverse 5'-CCATgATCTCTTCTACTCCTC CAATA-3') were cleaved with NcoI (bold) and EcoRV (underlined) and cloned into corresponding sites of pNG8048e [62], generating respectively pFtsKNter and pFtsKFL. The ClaI-XbaI fragment containing the *gfpmut1* gene of pSG1154 [63] was cloned into the corresponding sites of pFtsKNter and pFtsKFL yielding plasmids pKNterfp and pKFLgfp respectively. For fluorescence microscopy, cells from an overnight culture were diluted (1:100) into fresh medium and grown at 30 °C with agitation. At OD<sub>600</sub> = 0.5, supernatant of the nisin-producing strain NZ9700 [64] was added at dilution 1:1,000. At OD<sub>600</sub> = 1, 2 µl of culture was directly applied to a polysine microscope slide (Omnilabo, http://www1.omnilabo.nl) and covered with a cover glass. Cells were examined using a 100× oil immersion objective on a Zeiss microscope (Carl Zeiss, http://www. zeiss.com), and the fluorescent signal of GFP was detected using filter set 09 (excitation, 450-490 nm and emission, > 520 nm) from Zeiss. Images were captured with an Axion Vision camera (Axion Technologies, http://www.axiontech.com), and assembly of the final figures used Adobe Photoshop version 6.0.

## Supporting Information

**Figure S1.** Physical Analysis of *dif*<sub>SL</sub>-Mediated Integration Site in *L. lactis* Chromosome

Shown is Pulsed-Field gel Electrophoresis of *Smal*-digested chromosomes (A) and corresponding Southern hybridization (B) of WT strain (MG1363) and its *recA*-derivative (VEL1122) after *dif*-mediated integration of plasmids pCL235 or pCL237. Lanes: 1, MG1363; 2, MG1363::pCL235; 3, MG1363::pCL237; 4, VEL1122::pCL235; and 5, VEL1122::pCL237. (Electrophoresis conditions: 10 V/cm<sup>-1</sup>/13 h/15 s pulse time in Tris/borate/EDTA 0.05M). As predicted from the physical map of MG1363 chromosome [58] and genome sequence,

## References

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insertion of integrative plasmids at the *dif*<sub>SL</sub> site into the 610-kb *Smal* fragment generated two new *Smal* fragments of 350- and 260-kb in size. These corresponding restriction fragments were visualized in Southern hybridization using pCL235 and *iluD* gene as probes in the presence of ³²P-labeled λ DNA. A part of the chromosome population displayed a WT structure (i.e., with the plasmids pCL235 or pCL237 excised from the chromosome), an instability also observed when transforming *E. coli* strain with a nonreplicative plasmid containing the *E. coli* *dif* sequence (F. Cornet, personal communication).

Found at doi:10.1371/journal.pgen.0030117.sg001 (918 KB PDF).

**Figure S2.** Phylogenetic Analysis of the 42 Streptococcal Integrase Revealing Two Groups of Conserved Integrase, the XerS Group and the YnbA Group

Colors: red, integrases conserved in each streptococcal species; green, phage-related integrases; blue, transposon-related integrases; black, uncharacterized integrases. Bootstrap values are indicated for each branch.

Found at doi:10.1371/journal.pgen.0030117.sg002 (228 KB PDF).

**Table S1.** Bacterial Strains and Plasmids

Found at doi:10.1371/journal.pgen.0030117.st001 (28 KB PDF).

**Text S1.** Supporting Materials and Methods

Found at doi:10.1371/journal.pgen.0030117.sd001 (16 KB PDF).

## Accession Numbers

The National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov) accession number for *L. lactis* is NC_009004.

## Acknowledgments

The authors thank François-Xavier Barre, Sarah Bigot, Chantal Granadel, Bernard Martin, Jean-Pierre Claverys, and François Cornet for helpful discussions and the gift of plasmids and/or protocols. PLB particularly thanks Claude Bruand and Romain Mercier for useful suggestions in the *E. coli* experiments, Jonathan Filée for help in the phylogenetic analysis of streptococcal tyrosine recombinases, and François Cornet and Michael Chandler for insightful comments on the manuscript.

**Author contributions.** PLB and PR conceived and designed the experiments. PLB, MB, NC, MLDM, JL, DL, and TL performed the experiments. PLB, NC, and PR analyzed the data. PLB, MB, NC, MLDM, JL, DL, TL, and CP contributed reagents/materials/analysis tools. PLB, NC, MLDM, and PR wrote the paper.

**Funding.** All work in our laboratory is supported by the CNRS.

**Competing interests.** The authors have declared that no competing interests exist.

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<figure><img src="image_101.png" /></figure>
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<figure><img src="image_102.png" /></figure>
## Supporting Materials and Methods

### pGh9-derivative plasmids and *E. coli* strains constructions

pGh9-derivative plasmids were constructed as follows: relevant pairs of oligonucleotides containing *Xma*I linkers were annealed together, generating DNA duplexes with two *Xma*I-compatible ends. Only the left end of the duplexes recreated a full *Xma*I site, allowing further determination of its orientation by restriction analysis (only the synthetic sequences cloned in counterclockwise orientation were selected for experiments). These duplexes were 5'-phosphorylated and ligated with dephosphorylated *Xma*I-digested pCL52 or pGh9 vectors. The sequence of each cloned oligonucleotide was confirmed by DNA sequencing. The *dif*-Km<sup>R</sup>-*dif* cassette was introduced in *E. coli* strain LN2772 by integration-excision experiment [1], using pCL294 plasmid (Table S1). *E. coli* E359-derivative mutants carrying *xerC2::Ap*, *xerD::Ap*, or *ftsKc::Ap* were obtained by P1-mediated transduction using phage lysates kindly provided by F. Cornet. The *ftsKc::Ap* disruption corresponds to the replacement of *Fsp*I internal fragments of *ftsK* by the *bla* gene, and allows the synthesis of only the 316 first amino acids of *FtsK* (F. Cornet, personal communication). Allelic replacements were verified by PCR, and the *xer*<sup>-</sup> phenotype was tested by *in vivo* plasmid resolution assay using plasmid pFX142 [2]. The *recA56* allele was introduced by conjugation by co-transfer with *srl::Tn10* from strain JC10240 [3], and the *recA* phenotype was verified by UV-radiation sensitivity.

### Phylogenetic analysis of the streptococcal tyrosine recombinases

Tyrosine recombinase protein sequences were obtained from BLASTp searches (http://www.ncbi.nlm.nih.gov/sutils/genom_tree.cgi?) against ten streptococcal genomes (strains; *S. agalactiae* NEM316 and 2603V/R, *S. pneumoniae* TIGR4 and R6, *S. mutans*
UA159, *S. pyogenes* M1 GAS, M315, M8232, and SSI-1, and *L. lactis* IL1403), using *E. faecalis* CodV (XerC-like), *E. faecalis* RipX (XerD-like), and *S. pneumoniae* XerD (spr1692) sequences as queries. A total of 65 putative integrases were identified, and proteins from the same streptococcal species with >99% of amino acids identities were considered as duplicate and removed from the analysis. The remaining 42 non-redundant streptococcal integrases and the *E. faecalis* CodV and RipX sequences were aligned using CLUSTALX ver. 1.81 [4]. Phylogenic trees were constructed using PHYLIP 3.6 package [5]. The CLUSTALX alignment was run with the PROTDIST program with default settings to identify separation distances, and the calculated distances used as input file for NEIGHBOR program, with the *E. faecalis* CodV forced as outgroup. Bootstrap values were determined by performing SEQBOOT (500 repetitions) of the CLUSTALX alignment. The resulting file was used as input for PROTDIST program, with a 44 times randomization of the input order of sequences. A consensus tree was determined from the 500 best trees generated, and branches with bootstrap values <50% were collapsed together.

**References for Supporting Materials and Methods**

1. Corre J, Cornet F, Patte J, Louarn JM (1997) Unraveling a region-specific hyper-recombination phenomenon: genetic control and modalities of terminal recombination in *Escherichia coli*. *Genetics* 147: 979-989.

2. Aussel L, Barre FX, Aroyo M, Stasiak A, Stasiak AZ et al. (2002) FtsK is a DNA motor protein that activates chromosome dimer resolution by switching the catalytic state of the XerC and XerD recombinases. *Cell* 108: 195-205.
3. Capiaux H, Lesterlin C, Perals K, Louarn JM, Cornet F (2002) A dual role for the FtsK protein in *Escherichia coli* chromosome segregation. EMBO Rep 3: 532-536.

4. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25: 4876-4882.

5. Felsenstein J (1989) PHYLIP - Phylogeny Inference Package (Version 3.2). Cladistics 5: 164-166.
<figure><img src="image_103.png" /></figure>

A

<figure><img src="image_104.png" /></figure>

B
<figure><img src="image_105.png" /></figure>

0.1
<table><caption>TABLE S1. Bacterial strains and plasmids</caption><thead><tr><th>Plasmid or strain</th><th>Relevant properties</th><th>Origin</th></tr></thead><tbody><tr><td colspan="3"><strong>Plasmids</strong></td></tr><tr><td>pCL52</td><td>Em<sup>R</sup>, a 2.4-kb <i>E. coli</i> cloning vector derived from pBluescript KSII</td><td>[1]</td></tr><tr><td>PGh9</td><td>Em<sup>R</sup>, a 3.7-kb lactococcal <i>repA</i><sup>ts</sup> cloning vector</td><td>[2]</td></tr><tr><td>pFC23</td><td>Cm<sup>R</sup>, pKO3-derivative plasmid (<i>repA</i><sup>ts</sup>) containing the <i>rpsL</i><sup>+</sup> gene. Used for replacement of the <i>E. coli</i> <i>dif</i> site by allelic exchange</td><td>[3]</td></tr><tr><td>pFX170</td><td>Ap<sup>R</sup>, full-length <i>E. coli</i> FtsK protein fused to a FLAG epitope on its N-terminus and cloned into pBAD24 vector</td><td>[4]</td></tr><tr><td>pFX144</td><td>Cm<sup>R</sup>, pKO3-derivative (<i>repA</i><sup>ts</sup>) plasmid. Used for allelic exchange at <i>xerC</i> locus in <i>E. coli</i></td><td>[5]</td></tr><tr><td>pCL231</td><td>Em<sup>R</sup>, pGh9-derivative (<i>repA</i><sup>ts</sup>) plasmid. Contains a 37-bp fragment, <i>dif-2</i> (5'-TAACATCTTTCCgAAAAACTgTAA TTTTCTTgACAAT-3') cloned at the <i>XmaI</i> site (the 31-bp lactococcal <i>dif</i><sub>SL</sub> site is indicated in bold)</td><td>This work</td></tr><tr><td>pCL235</td><td>Em<sup>R</sup>, pCL52-derivative plasmid. Contains the same 37-bp synthetic fragment than pCL231</td><td>This work</td></tr><tr><td>pCL237</td><td>Em<sup>R</sup>, pCL52-derivative plasmid. Contains a 27-bp lactococcal <i>dif</i><sub>SL</sub> variant, <i>dif-8</i> (5'-CTTTCCgAAAAACT gTAATTTTCTTgA-3') of pCL233 (see Table 2) cloned at the <i>XmaI</i> site</td><td>This work</td></tr><tr><td>pCL263</td><td>Em<sup>R</sup>, 5.35-kb <i>XbaI-ClaI</i> fragment from pFX170 (containing</td><td>This work</td></tr></tbody></table>
<table>
  <tr>
    <td></td>
    <td>the FLAG-tagged full-length <i>ftsK</i> gene of <i>E. coli</i>, the araBAD promoter, and the <i>araC</i> gene) cloned at the corresponding sites of pCL52</td>
    <td></td>
  </tr>
  <tr>
    <td>pCL294</td>
    <td>Cm<sup>R</sup>, 2.04-kb <i>dif</i>-2-Km<sup>R</sup>-<i>dif</i>-2 cassette cloned at the <i>XmnI</i> site of pFC23. Used for introducing the cassette at the <i>E. coli</i> <i>dif</i> locus</td>
    <td>This work</td>
  </tr>
  <tr>
    <td>pCL297</td>
    <td>Cm<sup>R</sup>, lactococcal <i>xerS</i> ORF (<i>ymfD</i>) cloned into one <i>NcoI</i> site of pFX144. <i>xerS</i> is expressed from transcriptional read-through of the upstream <i>cat</i> gene</td>
    <td>This work</td>
  </tr>
  <tr>
    <td>pCL403</td>
    <td>Em<sup>R</sup>, pGh9-derivative (<i>repA</i><sup>ts</sup>) plasmid. Contains a 37-bp fragment (5'-TAACATCTTTCCgAAAAACTATAATTT TCTTgAAAAA-3') cloned at the <i>XmaI</i> site (the 31-bp pneumococcal <i>dif</i><sub>SL</sub> site is indicated in bold)</td>
    <td>This work</td>
  </tr>
  <tr>
    <td>pKNtergfp</td>
    <td>Em<sup>R</sup>, 181 first amino acids of the lactococcal FtsK protein fused to Gfpmut1 and expressed from P<sub>nisA</sub> of pNG8048e vector</td>
    <td>N. Campo, laboratory collection</td>
  </tr>
  <tr>
    <td>pKFLgfp</td>
    <td>Em<sup>R</sup>, full-length lactococcal FtsK protein fused to Gfpmut1 and expressed from P<sub>nisA</sub> of pNG8048e vector</td>
    <td>N. Campo, laboratory collection</td>
  </tr>
</table>

## Strains

### L. lactis

<table>
  <tr>
    <td>MG1363</td>
    <td>Wild Type strain. Plasmid-free derivative strain of NCDO712</td>
    <td>[6]</td>
  </tr>
  <tr>
    <td>VEL1122</td>
    <td>Tc<sup>R</sup>, MG1363 <i>recA::tetM</i></td>
    <td>[7]</td>
  </tr>
  <tr>
    <td>NZ9000</td>
    <td>MG1363 <i>pepN::nisRK</i>. Recipient strain for the nisin expression system.</td>
    <td>[8]</td>
  </tr>
</table>
S. pneumoniae

<table><tr><td>R800</td><td>Wild Type strain. Derivative of strain R6</td><td>[9]</td></tr><tr><td>S501</td><td>R800 <i>xerS</i> (spr1046::<i>Km</i>)</td><td>This work</td></tr><tr><td>S502</td><td>R800 <i>ftsK</i><sub>C</sub> (spr0781::<i>Km</i>)</td><td>This work</td></tr></table>

E. coli

<table><tr><td>LN2772</td><td>Str<sup>R</sup>, Tc<sup>R</sup>, W1485 <i>leu thyA deoB</i> or <i>C supE rpsL</i><br>Δ(<i>dif</i>)58::Tc</td><td>[3]</td></tr><tr><td>LN3038</td><td>Str<sup>R</sup>, Tc<sup>R</sup>, W1485 <i>leu thyA deoB</i> or <i>C supE rpsL lacZ::Tn10</i>.<br>This strain has a wild type phenotype for XerCD/<i>dif</i><br>recombination.</td><td>F. Cornet, laboratory<br>collection</td></tr><tr><td>E359</td><td>Str<sup>R</sup>, Km<sup>R</sup>, LN2772 with the lactococcal <i>dif</i>-2-Km<sup>R</sup>-<i>dif</i>-2<br>cassette inserted in the <i>tetA</i> gene</td><td>This work</td></tr><tr><td>E367</td><td>Str<sup>R</sup>, Tc<sup>R</sup>, Cm<sup>R</sup>, LN2772 containing pCL297</td><td>This work</td></tr><tr><td>E368</td><td>Str<sup>R</sup>, Cm<sup>R</sup>, E359 with one <i>dif</i><sub>SL</sub> site (the Km<sup>R</sup> cassette has<br>been excised by XerS/<i>dif</i><sub>SL</sub> recombination) and containing<br>pCL297</td><td>This work</td></tr><tr><td>E372</td><td>Str<sup>R</sup>, Km<sup>R</sup>, Ap<sup>R</sup>, E359 <i>ftsK</i><sub>C</sub>::Ap</td><td>This work</td></tr><tr><td>E375</td><td>Str<sup>R</sup>, Tc<sup>R</sup>, Cm<sup>R</sup>, LN3038 containing pCL297</td><td>This work</td></tr><tr><td>E378</td><td>Str<sup>R</sup>, Ap<sup>R</sup>, Cm<sup>R</sup>, E372 with one <i>dif</i><sub>SL</sub> site (the Km<sup>R</sup> cassette<br>has been excised by XerS/<i>dif</i><sub>SL</sub> recombination) and<br>containing pCL297</td><td>This work</td></tr><tr><td>E379</td><td>Str<sup>R</sup>, Km<sup>R</sup>, Tc<sup>R</sup>, E359 <i>recA56</i></td><td>This work</td></tr><tr><td>E408</td><td>Str<sup>R</sup>, Km<sup>R</sup>, Ap<sup>R</sup>, Tc<sup>R</sup>, E359 <i>xerC</i>::Ap</td><td>This work</td></tr><tr><td>E409</td><td>Str<sup>R</sup>, Km<sup>R</sup>, Ap<sup>R</sup>, Tc<sup>R</sup>, E359 <i>xerD</i>::Ap</td><td>This work</td></tr></table>
References for Table S1

1. Campo N, Daveran-Mingot M-L, Leenhouts KJ, Ritzenthaler P, Le Bourgeois P (2002) Cre/loxP recombination system for large genome rearrangements in *Lactococcus lactis*. Appl Environ Microbiol 68: 2359-2367.

2. Maguin E, Prévots H, Ehrlich SD, Gruss A (1996) Efficient insertional mutagenesis in *Lactococci* and other Gram-positive bacteria. J Bacteriol 178: 931-935.

3. Cornet F, Mortier I, Patte J, Louarn JM (1994) Plasmid pSC101 harbors a recombination site, *psi*, which is able to resolve plasmid multimers and to substitute for the analogous chromosomal *Escherichia coli* site *dif*. J Bacteriol 176: 3188-3195.

4. Yates J, Aroyo M, Sherratt DJ, Barre FX (2003) Species specificity in the activation of Xer recombination at *dif* by FtsK. Mol Microbiol 49: 241-249.

5. Aussel L, Barre FX, Aroyo M, Stasiak A, Stasiak AZ et al. (2002) FtsK is a DNA motor protein that activates chromosome dimer resolution by switching the catalytic state of the XerC and XerD recombinases. Cell 108: 195-205.

6. Gasson MJ (1983) Plasmid complements of *Streptococcus lactis* NCDO712 and other lactic *Streptococci* after protoplast-induced curing. J Bacteriol 154: 1-9.

7. Duwat P, Ehrlich SD, Gruss A (1995) The *recA* gene in *Lactococcus lactis*: characterization and involvement in oxydative and thermal stress. Mol Microbiol 17: 1121-1131.

8. Kuipers OP, de Ruyter PG, Kleerebezem M, de Vos WM (1998) Quorum sensing-controlled gene expression in lactic acid bacteria. J Biotechnol 64: 15-21.
9. Lefevre JC, Claverts JP, Sicard AM (1979) Donor deoxyribonucleic acid length and marker effect in pneumococcal transformation. J Bacteriol 138: 80-86.
# Are two better than one? Analysis of an FtsK/Xer recombination system that uses a single recombinase

Sophie Nolivos¹,², Carine Pages¹,², Philippe Rousseau¹,², Pascal Le Bourgeois¹,² and François Cornet¹,²,*

¹Laboratoire de Microbiologie et de Génétique Moléculaire, CNRS and ²Université de Toulouse, Université Paul Sabatier, F-31000, Toulouse, France

Received March 22, 2010; Revised May 18, 2010; Accepted May 19, 2010

## ABSTRACT

Bacteria harbouring circular chromosomes have a Xer site-specific recombination system that resolves chromosome dimers at division. In *Escherichia coli*, the activity of the XerCD/dif system is controlled and coupled with cell division by the FtsK DNA translocase. Most Xer systems, as XerCD/dif, include two different recombinases. However, some, as the *Lactococcus lactis* XerS/dif<sub>SL</sub> system, include only one recombinase. We investigated the functional effects of this difference by studying the XerS/dif<sub>SL</sub> system. XerS bound and recombined dif<sub>SL</sub> sites *in vitro*, both activities displaying asymmetric characteristics. Resolution of chromosome dimers by XerS/dif<sub>SL</sub> required translocation by division septum-borne FtsK. The translocase domain of *L. lactis* FtsK supported recombination by XerCD/dif, just as *E. coli* FtsK supports recombination by XerS/dif<sub>SL</sub>. Thus, the FtsK-dependent coupling of chromosome segregation with cell division extends to non-rod-shaped bacteria and outside the phylum Proteobacteria. Both the XerCD/dif and XerS/dif<sub>SL</sub> recombination systems require the control activities of the FtsKγ subdomain. However, FtsKγ activates recombination through different mechanisms in these two Xer systems. We show that FtsKγ alone activates XerCD/dif recombination. In contrast, both FtsKγ and the translocation motor are required to activate XerS/dif<sub>SL</sub> recombination. These findings have implications for the mechanisms by which FtsK activates recombination.

## INTRODUCTION

Most known bacteria have circular chromosomes. This circular form facilitates the termination of replication, but renders recombination-based repair hazardous, because sister chromatid exchanges between circles may result in dimer formation [reviewed in (1,2)]. Chromosome dimers cannot be segregated to daughter cells and must therefore be resolved, to generate monomers, before cell division. Xer site-specific recombination systems are responsible for this dimer resolution.

The resolution of chromosome dimers has been studied mostly in *Escherichia coli* [reviewed in (1,3,4)]. It involves two site-specific recombinases of the Y-recombinase family, XerC and XerD, acting at a recombination site, *dif*, located in the termination of replication region (Figure 1A). In contrast to the XerCD/dif system, most systems driven by Y recombinases, for example the Cre/loxP model system from plasmid P1, use a single recombinase (5). Recombination occurs in a complex containing four monomers of recombinase bound to two recombination sites synapsed in an antiparallel configuration (Figure 1B). A first pair of recombinases cuts and transfers a first pair of strands, producing a Holliday junction (HJ)-containing intermediate. Subsequent catalysis by the second pair of recombinases resolves this intermediate to generate recombinant molecules. In the XerCD/dif system, the synaptic complex consists of two XerC and two XerD monomers bound to two *dif* sites. The *dif* site contains binding sites for XerC and XerD separated by a 6-bp central region (CR), at the edges of which, strand exchanges are catalysed (Figure 1A). XerC or XerD may catalyse the exchange of the first pair of strands, depending on external controls imposed on the synaptic complex (6,7). *In vivo* and *in vitro* studies have shown that the XerCD/dif complex naturally tends to

<footnote>*To whom correspondence should be addressed. Tel: +33 561 335 985; Fax: +33 561 335 886; Email: francois.cornet@ibcg.biotoul.fr</footnote>
<figure><img src="image_106.png" /><figcaption>**Figure 1.** The FtsK-XerCD/dif and FtsK-XerS/dif<sub>SL</sub> systems. (A) Left: diagram of the XerC, XerD, XerS and Cre proteins, with their length and domain organization (in amino acid). The conserved residues involved in catalysis are indicated. Right: *dif*<sub>SL</sub> and *dif* sites of representative bacteria with the recombinase binding sites, separated by the central region (CR) indicated. In *dif*<sub>SL</sub> sites, upper case bases are part of the previously defined minimal site (28) and bases shown in bold typeface are inverted repeats within this minimal site. The left-half site is indicated by the pale grey bar and the right-half site by the dark grey bar. This convention is used throughout the paper. In *dif* sites, the two half sites recognized by XerC and XerD, respectively, are indicated. *Lla*, *L. lactis*; *Spn*, *Streptococcus pneumoniae*; *Spy*, *S. pyogenes*; *Sag*, *S. agalactiae*; *Eco*, *E. coli*; *Hin*, *H. influenzae*; *Vch*, *V. cholerae*; *Ngo*, *Neisseria gonorrhoeae*; *Bsub*, *B. subtilis*. (B) A diagram of the mechanism of recombination by Y-recombinase based on the data obtained for the Cre/loxP system. Y indicates the catalytic tyrosine residue. OH is the 5' hydroxyl group created by DNA cleavages.</figcaption></figure>

initiate recombination by XerC-mediated strand exchange (7). This bias is thought to be due to both the asymmetry of the *dif* sequence and the use of two different recombinases. This “XerC-first” pattern applies to activities of XerCD other than the resolution of chromosome dimers: during the resolution of plasmid multimers at the *psi* or *cer* sites (8) and during the integration of bacteriophage genomes into their host *dif* sites (9). In these cases, HJ resolution by XerD catalysis is either not required (*cer* site and phage integration) or is induced by isomerization of the HJ intermediate (*psi* site, Figure 1B). In chromosomal XerCD/*dif* complexes, recombination is controlled by FtsK, a DNA translocase associated with the cell division septum (3). In the absence of FtsK, XerD has no catalytic activity and HJ-intermediates may be generated by XerC catalysis. However, these intermediates are not fully resolved to generate products, but are instead reconverted into substrates, by further XerC catalysis (7,10). Chromosome dimer resolution requires FtsK, which induces recombination by forcing XerD to catalyse the exchange of the first pair of strands.

FtsK is required for both cell division and faithful
chromosome segregation [reviewed in (3)]. Its N-terminal
domain is essential for growth and contains

transmembrane helices that target FtsK to the division
septum. Its C-terminal domain (FtsK<sub>C</sub>), which is dispens-
able for viability, encodes the translocation motor (7). The
α and β subdomains of FtsK<sub>C</sub> carry Walker-type ATPase
motifs and form a hexameric motor (11). The extreme
C-terminal subdomain, FtsKγ, controls FtsK transloca-
tion and Xer recombination. FtsKγ contains a
DNA-binding motif that binds to specific DNA motifs,
the KOPS (5'-GGGNAGGG). KOPSs are preferentially
oriented towards the *dif* site, thereby orienting transloca-
tion towards *dif* (12–17). Translocation stops when the
XerCD/*dif* complex is reached, and this termination of
translocation is FtsKγ-independent (18). Recombination
is then induced by a mechanism thought to involve a par-
ticular interaction of FtsK<sub>C</sub> with the DNA in the imme-
diate vicinity of *dif* (19) and a direct interaction between
FtsKγ and XerD (20). This second interaction induces
XerD-mediated strand exchange *in vitro*, suggesting it
induces a recombination pathway in which XerD catalyses
the exchange of the first pair of strands, leading to pro-
ductive recombination and dimer resolution (20).

Homologs of FtsK are found in most bacteria, in which
they are involved in DNA transfer during conjugation,
sporulation and chromosome segregation (21–25).