FtsK-Dependent Dimer Resolution on Multiple Chromosomes in the Pathogen Vibrio cholerae

Unlike most bacteria, Vibrio cholerae harbors two distinct, nonhomologous circular chromosomes (chromosome I and II). Many features of chromosome II are plasmid-like, which raised questions concerning its chromosomal nature. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle, further calling into question the mechanisms ensuring the synchronous management of chromosome I and II. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In Escherichia coli, chromosome dimers are resolved by the addition of a crossover at a specific site, dif, by two tyrosine recombinases, XerC and XerD. The process is coordinated with cell division through the activity of a DNA translocase, FtsK. Many E. coli plasmids also use XerCD for dimer resolution. However, the process is FtsK-independent. The two chromosomes of the V. cholerae N16961 strain carry divergent dimer resolution sites, dif1 and dif2. Here, we show that V. cholerae FtsK controls the addition of a crossover at dif1 and dif2 by a common pair of Xer recombinases. In addition, we show that specific DNA motifs dictate its orientation of translocation, the distribution of these motifs on chromosome I and chromosome II supporting the idea that FtsK translocation serves to bring together the resolution sites carried by a dimer at the time of cell division. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution with cell division for each of the two V. cholerae chromosomes. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.


Introduction
Vibrio cholerae, the causative agent of cholera, harbors two nonhomologous circular chromosomes [1]. The majority of genes believed to be necessary for the basic life processes of V. cholerae are carried on the 2.96 Mbp chromosome I, whereas the 1.07 Mbp chromosome II only harbors a few essential genes [1]. The preferential transcription of genes from chromosome II during colon colonization [2] suggests that this genomic organization is important for pathogenicity. Likewise, other bacteria with multiple chromosomes can adopt several different life cycles [3], which led to the idea that multipartite genomes offer a selective advantage for the adaptation to very different environmental conditions. Nevertheless, most bacteria harbor a single chromosome. In contrast, there is no apparent limit to the size and numbers of chromosomes harbored by eukaryotic cells. An important difference between bacteria and eukaryotes is that specific machineries appear to exist for the coordinated maintenance of each chromosome of a given bacterium, whereas eukaryotic cells possess a single global system for all chromosomes [4][5][6][7][8][9][10][11][12]. For instance, the two V. cholerae chromosomes harbor different partition systems [4,7] and initiation of their replication is governed by different mechanisms [6,8,9]. In addition, many features of V. cholerae chromosome II, such as its partition system, are plasmid-like, which raised questions concerning its chromosomal nature [1,10,13]. Plasmid replication and segregation are generally not coordinated with the bacterial cell cycle [14], further raising questions on the mechanisms ensuring the synchronous management of chromosome I and II.
A second major difference between bacteria and eukaryotes is intrinsic to the structure of chromosomes: in bacteria, chromosomes are generally covalently closed circular DNA molecules while they are linear in eukaryotes. DNA circularity can result in the formation of chromosome dimers by homologous recombination [15], which poses a barrier to the segregation of genetic information if they are not resolved before cell division ( Figure 1A). Indeed, inactivation of chromosome dimer resolution (CDR) in Escherichia coli results in ,15% cell death per generation under laboratory growth conditions [16], which corresponds to the estimated rate of chromosome dimers formed at each cell generation [17]. This prompted us to study how dimer resolution is achieved on each of the two V. cholerae chromosomes.
The mechanism of CDR was originally elucidated in E. coli. In this organism, it depends on the addition of a crossover at dif, a 28bp site located at the opposite of the origin of replication on the chromosome, by two related tyrosine recombinases, XerC and XerD ( Figure 1A; see [18] for a review). In addition, CDR depends on two activities of a cell division protein, FtsK. First, FtsK functions as a DNA pump anchored in the septum [19,20]. It loads on DNA trapped within the division septum due to dimer formation ( Figure 1A). FtsK loading is oriented by specific DNA motifs, the KOPS, which dictates the orientation of translocation ( Figure 1A; [21]). KOPS are skewed on the two replichores of the chromosome with dif located at the junction of their polarity [22,23]. Thus, dif sites carried by a dimer are brought together by FtsK translocation ( Figure 1A). Second, FtsK serves to activate recombination at dif via a direct interaction with XerD [24,25]. dif contains two 11bp binding sites for XerC and XerD, separated by a central region at the outer boundary of which recombination occurs. The interaction between XerD and FtsK allows XerD to perform a first pair of strand exchanges [20], resulting in the formation of a Holliday junction (HJ). This HJ is converted to a crossover by a second pair of strand exchanges, which is catalyzed by XerC independently of FtsK ( Figure 1B, chromosomal pathway). Thus, in E. coli, the requirement for FtsK to bring dif sites together and to activate the catalytic activity of XerD permits coordination of CDR with the last stage of cell division [26].
The E. coli pathway of CDR is not universal. For instance, Streptococci and Lactococci possess only a single tyrosine recombinase, XerS, for CDR [27]. Plasmid and viruses have also adopted different site-specific recombination systems to avoid multimerization of their genome. In E. coli, some of them depend on their own recombinases, such as phage P1, which encodes the Cre tyrosine recombinase [28], while others use the two Xer recombinases of their host [29,30]. In the later case, XerC-catalysis initiates recombination independently of FtsK ( Figure 1B, plasmid pathway, [18]). In this case, however, recombination requires ,200bp of accessory sequences flanking the plasmid sites and which are bound by accessory proteins.
Orthologues of E. coli xerC, xerD and ftsK are readily identified on the larger chromosome of the V. cholerae strain N16961 (xerC Vc , xerD Vc , ftsK Vc , Figure S1) whereas its second chromosome does not encode any site-specific recombination system that could be implicated in CDR apart from the superintegron integrase (IntIA, Figure S1). N16961 chromosome I and II both carry dif-like sequences, dif1 and dif2, which were originally identified as integration sites for the Cholera Toxin phage, CTXw [31]. The weak filamentous phenotype of V. cholerae cells deleted for xerC or dif1 fits with a defect in CDR [31]. However, two features of the V. cholerae Xer recombination system, which could be linked to the coexistence of distinct, non-homologous chromosomes inside the same bacterium, were intriguing. First, dif2 differs from the dif consensus of c-Proteobacteria by 5 bases, four of which belong to the central region ( Figure 1B). Such a divergence is only found on plasmid sites, which, coupled with the other plasmid-like features of chromosome II, suggested that chromosome II dimer resolution might follow a plasmid pathway. Second, it was reported that the position of cleavage of XerD Vc on dif1 might differ from the one of its E. coli orthologue on dif [32], even if dif1 differs from the dif consensus of c-Proteobacteria by only 2 bases ( Figure 1B), further raising questions on the exact mechanisms coordinating CDR of chromosome I and II with the cell cycle.
Here, we present the first formal study of CDR in V. cholerae and measure the rate of chromosome dimer formation on its two chromosomes under laboratory growth conditions. We show that the cell division protein FtsK Vc is required for recombination by XerC Vc and XerD Vc at dif1 and dif2. In addition, we show that the activity of FtsK Vc is directed by specific DNA motifs, which display the same skewed distribution on the two chromosomes, dif1 and dif2 being located at the junction of their polarity. Taken together, these results suggest that the same FtsK-dependent mechanism coordinates dimer resolution on each of the two V. cholerae chromosomes with cell division. Chromosome II dimer resolution thus stands as a bona fide chromosomal process.

Chromosome Dimer Formation in V. cholerae
The growth of V. cholerae strains deficient in CDR was directly compared to the growth of their parental strain in competition experiments in rich media ( Figure 2). These experiments revealed a defect of 5.8% and 3% per cell per generation for Ddif1 and Ddif2 cells, respectively, compared to their wild type counterparts. Since these growth defects were entirely suppressed in a recA background (Figure 2), they directly reflect the rates of dimer formation on chromosome I and II, f dimer Chr1 and f dimer Chr2 (See Material and Methods). The 8.6% growth defect of xerC Vc cells, which was also suppressed in a recA background, reflects the total rate of chromosome dimer formation in V. cholerae, f dimer Chr1+2 ( Figure 2). Interestingly, f dimer Chr1+2 equals 12(12f dimer Chr1 )(12 f dimer Chr2 ), indicating that dimer formation on the two V. cholerae chromosomes is independent.
In Vitro Cleavage by the V. cholerae recombinases on dif1 and dif2 Recombinase-mediated strand cleavage can be assayed in vitro using suicide substrates that contain a nick opposite of the position of cleavage ( Figure 3A). Cleavage of the continuous strand of a suicide substrate generates a double strand break that prevents re-ligation ( Figure 3B). This leads to (i) the accumulation of covalent protein/ DNA complexes between the attacking recombinase and the 59-end fragment of the continuous strand and (ii) the accumulation of free 39end fragments of the continuous strand ( Figure 3B). XerC Ec and XerD Ec each cleave a specific strand on dif Ec . The strand cleaved by XerC Ec is termed Top strand. The strand cleaved by XerD Ec is termed Bottom strand. Following this convention, suicide substrates in which the continuous strand is expected to be cleaved by XerC Vc are called Top strand suicide substrates and suicide substrates in which the continuous strand is expected to be cleaved by XerD Vc are called Bottom strand suicide substrates ( Figure 3A).
Labeling the 59-end of the continuous strand of suicide substrates allows the detection of covalent recombinase/DNA

Author Summary
During proliferation, DNA synthesis, chromosome segregation, and cell division must be coordinated to ensure the stable inheritance of the genetic material. In eukaryotes, this is achieved by checkpoint mechanisms that delay certain steps until others are completed. No such temporal separation exists in bacteria, which can undergo overlapping replication cycles. The eukaryotic cell cycle is particularly well suited to the management of multiple chromosomes, with the same replication initiation and segregation machineries operating on all the chromosomes, while the bacterial cell cycle is linked to genomes of less complexity, most bacteria harboring a single chromosome. The discovery of bacteria harboring multiple circular chromosomes, such as V. cholerae, raised therefore a considerable interest for the mechanisms ensuring the synchronous management of different replicons. Here, we took advantage of our knowledge of chromosome dimer resolution, the only bacterial segregation process for which coordination with cell division is well understood, to investigate one of the mechanisms ensuring the synchronous management of the smaller, plasmid-like, and larger, chromosome-like, replicons of V. cholerae. complexes ( Figure 3C). The molecular weight of XerC Vc and XerD Vc being very similar, we used a maltose binding protein fusion of XerC Vc (MBPXerC Vc ) in conjunction with XerD Vc to avoid any confusion between the two possible covalent complexes. For both dif1 and dif2, MBPXerC Vc -DNA covalent complexes accumulated when Top strand suicide substrates were used ( Figure 3C, T1 and T2, respectively), indicating that XerC Vc cleaves the Top strands of dif1 and dif2. Furthermore, XerD Vc -DNA covalent complexes accumulated when Bottom strand suicide substrates were used ( Figure 3C, B1 and B2), indicating that XerD cleaves the bottom strands of dif1 and dif2.
The position of cleavage of XerC Vc and XerD Vc were then determined by comparing of the length of the free DNA fragments liberated by recombinase cleavage to a ladder obtained by chemical cleavage at purine bases of the suicide substrates ( Figure 3D). To this aim, the continuous strands of the suicide substrates were labeled on their 39 end. Cleavage by tyrosine recombinases generates a 59OH DNA extremity whereas chemical cleavage leaves a 59 phosphate. As a consequence, the free DNA fragments had to be first phosphorylated by kinase treatment ( Figure 3D, PNK) in order to be compared with the chemical cleavage ladder ( Figure 3D, G+A). We thus found that XerC Vc and XerD Vc cleave DNA at the junction between their respective binding site and the central region of dif1 and dif2 ( Figure 3D, black arrows).

FtsK-Dependent Recombination at dif1 and dif2
Analysis of the DNA sequence immediately upstream and downstream of dif1 and dif2 in different Vibrio species did not reveal any conserved motifs that could serve to bind accessory proteins (data not shown). FtsK Vc was thus left as the most likely candidate for activation of Xer recombination at both sites. To test this possibility, we reconstituted the V. cholerae Xer system in E. coli cells deleted for their natural FtsK/XerCD system. We used a xerC and xerD E. coli strain, which was also ftsK C 2 . This strain produces only the N-terminal domain of FtsK Ec , essential for viability [33], but lacks production of the C-terminal domain of FtsK Ec , which is necessary for recombination at dif Ec [34]. XerC Vc was expressed in conjunction with XerD Vc from the chromosomal E. coli xerC promoter. The production of FtsK Vc was controlled by placing the full length ftsK Vc ORF under an arabinose-inducible promoter on a high-copy number plasmid. A low-copy plasmid carrying two recombination sites in direct repeats was used as a reporter. Recombination between the two repeated sites results in the excision of the intervening DNA, which can be monitored by agarose gel electrophoresis. For both dif1 and dif2, the amount of recombination correlated with the amount of arabinose used for induction, indicating that Xer recombination at dif1 and dif2 depends on FtsK Vc ( Figure 4A).
To determine the order of the strand exchanges in the recombination reactions, we monitored plasmid recombination in a set of four strains encoding either wild-type XerC Vc and XerD Vc or the XerC YF Vc and XerD YF Vc mutants, in which the catalytic tyrosine is replaced by a phenylalanine ( Figure 4B , indicating that XerD Vc mediates the first pair of strand exchanges during both dif1 and dif2-recombination. Recombination products were likely still observed in XerC YF Vc XerD Vc cells since other cellular processes than Xer recombination are capable of resolving HJs [18]. However, the amount of product was considerably decreased, indicating that intermediate HJs are preferentially resolved to crossovers by the action of XerC Vc .
All together, these results indicate that FtsK Vc activates recombination at dif1 and dif2 by promoting the exchange of a first pair of strands by XerD Vc .

Species-Specificity in Xer Recombination Activation
Several residues implicated in the interaction between E. coli XerD and FtsK have been mapped [24,25]. These residues are not entirely conserved between the V. cholerae and E. coli proteins ( Figure 5A), suggesting that the interactions between the translocase and the recombinases might be specific in these two species. Nevertheless, both FtsK Ec and FtsK Vc could activate recombination by XerCD Ec and XerCD Vc at dif Ec , dif1 and dif2 ( Figure 5B).
However, the efficiency of recombination varied for each site and for each pairing of translocase/recombinases. XerCD Ec -recombination at dif Ec and dif1 reached 80% of efficiency whether FtsK Ec or FtsK Vc were produced ( Figure 5B, XerCD Ec , dif1 and dif Ec ). In contrast, XerCD Ec -recombination at dif2 was more efficient when activated by FtsK Ec than FtsK Vc ( Figure 5B, XerCD Ec , dif2). In addition, it did not reach 80% efficiency, even in the presence of the cognate partner translocase, FtsK Ec . XerCD Vc -recombination at dif Ec , dif1 and dif2 reached 80% of efficiency ( Figure 5B, XerCD Vc ). However, this required the presence of FtsK Vc . XerCD Vc -recombination at dif2 even fell below 20% when activated by FtsK Ec . Thus, the effect of species-specificity is more pronounced on dif2 than on dif1.

Importance of the Sequence of the Resolution Sites for the Stringent Control of Xer Recombination
We noticed that the V. cholerae recombinases could promote recombination between dif1 sites in the absence of FtsK production, albeit to a very low level ( Figure 5B, XerCD Vc , dif1, No FtsK). This was further exemplified on dif Ec substrates, in which 53% of recombination was observed without FtsK expression ( Figure 5B, XerCD Vc , dif Ec , No FtsK). Resolution products were detected in the absence of XerC catalysis ( Figure 5C, XerC YF Vc strains) but not in the absence of XerD catalysis ( Figure 5C, XerD YF Vc strains), signifying that XerD Vc catalysis initiated recombination. dif1 differs from the c-Proteobacteria consensus by only 2 bp, the substitution of A 17 by G and the substitution of A 10 by T ( Figure 5D). We therefore analyzed FtsK-independent XerCD Vc recombination at hybrid sites between dif, dif1 and dif2 to identify residues important for the above observation ( Figure 5D 10 increased FtsK-independent recombination to a level equivalent to dif Ec -recombination (dif15). In addition, when T 10 was altered to A in dif2, we observed a faint recombination product (dif23), which was significant since FtsK-independent recombination was never observed at dif2. Thus, G 17    To test for the ability of FtsK Vc to specifically recognize one of the E. coli KOPS motifs, we compared the efficiency with which it activates E. coli Xer recombination between plasmid-borne dif Ec sites flanked or not by the 59-GGGCAGGG-39 motif in an orientation that should prevent it from translocating towards dif Ec ( Figure 6B, KOPS-2 and KOPS-0, respectively). Here we observed that the efficiency of recombination dropped significantly on KOPS-2 when FtsK Ec or FtsK Vc were used as activators ( Figure 6B, FtsK Ec and FtsK Vc ). We then engineered an allele of ftsK Vc carrying identical mutations to the one shown to abrogate KOPS recognition in FtsK Ec [35]. No difference in recombination efficiency was noticeable between KOPS-0 and KOPS-2 when using this allele or its E. coli homologue ( Figure 6B; FtsK 50C Ec [N1296A; R1300A; E1303A] and FtsK Vc [N926A; R930A; E933A]). We conclude that FtsK Vc directly recognizes the GGGCAGGG motif and that recognition engages amino acids N926, R930 and E933.
We decided therefore to analyze the skew and frequency of the GGGCAGGG motif on chromosome I and II. GGGCAGGG is highly polarized on both chromosomes with statistically significant skews ( Figure 6C). On chromosome I, the skew switches precisely at dif1 whereas on chromosome II one motif is present on the reverse orientation a few kb before dif2 ( Figure 6C). However, it has been shown in E. coli that a single non-permissive KOPS motif in the vicinity of dif is not sufficient to impair recombination [23]. The frequency of GGGCAGGG is low on both V. cholerae chromosomes ( Figure 6C), suggesting that this motif is not sufficient by itself to provide polar orientation of FtsK Vc . We therefore analyzed the distribution of all octamers motif families with one degenerated position on both chromosomes. We ranked potential candidates according to their skew significance keeping only families that had a skew of at least 80% and a frequency of at least once every 30 kb. Only one family (GGGNAGGG) was among the 10 best candidates of both chromosomes. This family is highly skewed, frequent ( Figure 6C) and contains the experimentally active GGGCAGGG motif. Taken together, these results suggest that the GGGNAGGG motifs might function as KOPS in V. cholerae.

A Common Cell Division-Coordination Mechanism for Dimer Resolution of the Two V. cholerae Chromosomes
The strand exchanges catalyzed by XerC Vc and XerD Vc occur at the junction between their respective binding site and the central region of dif1 and dif2, as previously reported for the E. coli recombinases on dif (Figure 3). FtsK Vc promotes recombination at both sites by activating a first pair of strand exchanges mediated by XerD Vc (Figure 4), thanks to a species-specific interaction with the recombinases (Figure 5). In addition, GGGNAGGG motifs seem to function as FtsK Vc -Orienting Polar Sequences, their frequency and distribution on the two V. cholerae chromosomes suggesting that the FtsK Vc -translocase activity helps bring CDR sites together when dimers are formed on chromosome I or on chromosome II ( Figure 6). We conclude that the same FtsK-dependent mechanism controls dimer resolution on each of the two V. cholerae chromosomes. We have previously shown in E. coli that the requirement for FtsK to activate Xer recombination delays CDR to the time of septum closure [26], which is likely to also hold true in V. cholerae. Thus, the study of CDR provides the first example of a cell cycle coordination mechanism shared by the two V. cholerae chromosomes, which is similar to the way chromosomal maintenance processes are coordinated within the cell cycle of eukaryotes.

Dimer Formation Is Linked to Replicon Size
Many bacteria harbor multiple chromosomes, which seems an important determinant of their individual life styles. A few bacterial species harbor linear replicons in addition to circular, such as Agrobacterium tumefaciens and the Borrelia species [3]. In the vast majority of cases, however, the multiple chromosomes harbored within a bacterium are circular. Maintenance of circular replicons requires the resolution of dimers created by homologous recombination events. In V. cholerae, 5.8% of dimers per cell per generation are formed on the 2.96 Mbp chromosome I and 3% of dimers are created on the 1.07 Mbp chromosome II (Figure 2). Under similar growth conditions, 15.6% of dimers are generated on the 4.6Mbp E. coli chromosome (Figure 6). These results suggest that dimer formation increases with replicon size, possibly reaching a theoretical upper limit of 50% for very large replicons. In addition, the rate of dimer formation seems to vary exponentially with replicon size for small replicons. Based on this hypothesis, the frequency of chromosome dimer formation in V. cholerae would be 11% per cell generation if it carried a single circular chromosome of 4.03Mbp. Instead, we measured a total rate of 8.6% for the two chromosomes ( Figure 2). Thus, the particular genomic organization of the Vibrios seems to minimize chances for chromosome dimer formation, which is theoretically beneficial.

Generalization to Other Bacteria with Multiple Chromosomes
Putative dif sites were readily identified on each of the two chromosomes harbored by 7 additional c-Proteobacteria (Figure 7 and Figure S2). To determine dif sites in band a-Proteobacteria, we generated a profile Hidden Markov Model (HMM) based on the alignment of the putative CDR sites found in the larger chromosome of 27 c-Proteobacteria using the program HHMER. We then compared each sequence by hand to ensure the proper 6 bp spacing between the putative XerC and XerD binding sites. Putative dif sites were thus identified on each of the multiple chromosomes harbored by 10 b-Proteobacteria species and 5 a-Proteobacteria species (Figure 7 and Figure S3 and S4). A single pair of recombinases orthologous to XerC and XerD was found in each of the 22 additional c-, band a-Proteobacteria harboring multiple chromosomes, suggesting that a single pair of recombinases ensures dimer resolution of each of their non-homologous chromosomes. FtsK orthologues were also found. In addition, putative dif sites fell within 10 kb of the GC-skew inflection point (data not shown), suggesting that dimer resolution is under the  control of an FtsK-like homologue in all these species. Thus, the adoption of an FtsK-dependent dimer resolution system could be a key evolutionary step in the maintenance of large circular replicons.
Tuning of V. cholerae CDR to Achieve Efficient Recombination on Divergent dif Sites The sequence of Xer target sites, and especially of their central region, is a crucial determinant in the outcome of recombination [36,37]. Indeed, the central region of dif sites found in Proteobacteria with a single chromosome showed a high degree of conservation, most band c-Proteobacteria harboring a 'canonical' 59-TGTATA-39 motif (Figure 7 and Figure S2 and S3), suggesting that there is a selective pressure on the sequence of the dif central region. This is further illustrated by the lower recombination efficiency of the E. coli system on dif2 compared to dif1 ( Figure 5). In this regard, the V. cholerae Xer recombination system is remarkable since identical recombination efficiencies were obtained with the same pair of recombinases on dif1 and dif2 (Figure 4 and 5). However, XerCD Vc -mediated recombination at dif2 required a tighter interaction between the recombinases and their partner translocase than at dif1, since FtsK Ec promoted 50% of recombination at dif1 but less than 20% at dif2 ( Figure 5B, XerCD Vc , FtsK Ec ). In addition, a few alterations in the sequence of dif1 and dif2 decreased the stringency of the control exerted by FtsK Vc (Figure 5D), highlighting the extremely fine tuning of the different components of the V. cholerae CDR system.

Non-Homologous Chromosomes Carry dif Sites with Divergent Central Regions
We observed that in Proteobacteria with multiple chromosomes, the central regions of dif sites from non-homologous chromosomes are divergent, as in V. cholerae (Figure 7 and Figure  S2, S3, S4). A single exception was found in Burkholderia xenovarans, in which two of the three chromosomes of the bacterium harbor a resolution site with an identical central region. We reasoned therefore that some selective pressure imposes the divergence of the central regions of CDR sites carried by the different, nonhomologous chromosomes of bacteria with multipartite genomes, which competes with the selective pressure for dif central regions to adopt the preferential 59-TGTATA-39 motif. Indeed, the presence of dif sites with identical central regions on two non-homologous chromosomes could lead to the formation of chromosome fusions by Xer recombination, which would disrupt the selective advantage brought by the multipartite genomic organization. In support of this hypothesis, preliminary experiments indicate that harmonization of the two V. cholerae dif sites leads to chromosomal fusions (Val and Barre, unpublished observations). We are currently investigating how these fusions are formed and the consequences of harboring identical dif sites on separate chromosomes.

Strains, Plasmids, and Media
All growth experiments were done in LB-Lennox. Strains and plasmids are listed in Text S1. Briefly, V. cholerae strains were derived from N16961 [1] by allele exchange using pDS132 derivatives [38] and E. coli b2163 as a donor strain [39]. E. coli strains used for in vivo plasmid resolution assays and for growth competition were engineered as previously described in [25,40]. Mutations were confirmed by PCR and sequencing.

Growth Competition Assay
For growth competitions, E. coli cells were grown at 37uC with a 10006 dilution in fresh media every 12h [40]. Because of their higher growth rate, V. cholerae cells were grown at 30uC with a 100006 dilution every 12h. The numbers of CFU of mutated and parental cells in the cultures were determined by plating on cognate antibiotic plates every 12 or 24h, depending on the mutant growth defect. These numbers were used to calculate the number of generation of the parent cells between each time points substrate with triple overlapping GGGCAGGG sequences in the non-permissive orientation on both sides of the two dif sites. C. Scheme of the two V. cholerae chromosomes showing the distributions of the GGGCAGGG and GGGNAGGG motifs. Upper bars: motifs found in the leading strand; Lower bars: motifs found in the lagging strand. Number, frequency, skew and skew significance (p-skew) are indicated for each motif. Recently acquired genomic regions are indicated (superintegron, CTX and TLC prophages and the Vibrio Pathogenicity Island VPI). doi:10.1371/journal.pgen.1000201.g006 and the CFU ratio of mutated versus parent cells at each time point. This ratio varies exponentially with the number of generations. The proportion of cells that the mutant strain fails to produce at each doubling time of its parent is deduced from the coefficient of this exponential. This ratio is a good estimation of the rate of dimer formation (Text S1).

In Vitro Xer Assays
V. cholerae MBP-XerD and MBP-XerC recombinases were purified using nickel, amylose and heparin columns. The MBP tag was removed by thrombin digestion. dif1 and dif2 synthetic suicide substrates (Text S1) were obtained by annealing synthetic oligonucleotides purified by PAGE. 59-end labeling of oligonucleotides was performed using T4 DNA polynucleotide kinase and [ 32 P] c-ATP and 39end labeling using terminal transferase and [ 32 P] a-ddATP. Reactions were performed in 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1mM EDTA, 1 mg/ml of BSA, 40% glycerol and 0.2 pmol of radiolabeled probe for 2 hours at 37uC. Covalent complexes were analyzed by 12% SDS-PAGE and cleavage sites by 12% urea-PAGE. Radioactivity was detected on a STORM (GE Healthcare).

In Vivo Plasmid Resolution Assays
E. coli cells were transformed with the FtsK expression vector and then with the Xer recombination reporter plasmid, as described in [25]. 10 transformant colonies were pooled in 1 ml of LB, diluted 1006 in LB and grown to 0.6 OD at 37uC. Cells were then grown for an extra 2 hours at 37uC in the presence of 0.5% arabinose to induce FtsK production, unless otherwise indicated. Plasmid DNA was hydrolyzed with NdeI (single cutter). Recombination efficiency was computed as the amount of replicative product over the sum of the amount of substrate and of replicative product, which were separated by agarose gel electrophoresis and detected with SybrGreen staining using a LAS-3000 (Fuji Life Science).

Bioinformatics Analysis of Motifs Distribution
Leading strands were defined as the DNA strand reported in Genbank files downstream of the replication origin up to the terminus and the reverse complement strand from the terminus to the origin. The terminus position was chosen as the first nucleotide of the CDR site. Skew statistical significance was assessed by calculating the probability that the observed skew occurred by chance taking into account the fact that G-rich motifs are likely to be more frequent on the leading strand because of GC skew, as previously described [41]. Analysis on chromosome II was performed on a chimeric chromosome where the superintegron has been removed because this element carries more than 100 repetitions of the attC integration site, which hides the signal provided by octamer motifs. Figure S1 XerCD and FtsK tree.