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 Δdif1 and Δdif2 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 1−(1−f_dimer^Chr1)(1−f_dimer^Chr2), indicating that dimer formation on the two V. cholerae chromosomes is independent.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 2. Growth competition of V. cholerae deficient in CDR strains against their parent.

f: frequency of cells that the mutant strains fail to produce at each generation compared to their parent.

https://doi.org/10.1371/journal.pgen.1000201.g002

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 5′-end fragment of the continuous strand and (ii) the accumulation of free 3′-end 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).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 3. In vitro cleavage of dif1 and dif2 by the V. cholerae recombinases.

A. Putative XerC^Vc and XerD^Vc cleavage sites on dif1 and dif2 and scheme of the suicide substrates used in this study. The top and bottom strands of dif1 and dif2 are depicted as black and grey strands. Their equivalents in dif^Ec are cleaved by XerC^Ec and XerD^Ec, respectively. Grey triangles further indicate the positions equivalent to these where XerC^Ec and XerD^Ec cleave dif^Ec. A white triangle indicates the XerD^Vc-cleavage position reported for dif1 [32]. Top and bottom strand suicide substrates contain a nick opposite the position expected to be cleaved by XerC^Vc and XerDVc if the E. coli paradigm is followed, respectively. T1, B1, T2, B2: suicide substrates on dif1 and dif2, respectively. B. Scheme of a XerC-suicide cleavage reaction. C. Covalent complex formation by MBPXerC^Vc and XerD^Vc on suicide substrates. Schemes of substrates and products are shown on the top and on the right of the gel, respectively. Suicide substrates were labeled on the 5′ side of the continuous strand, as indicated (5′*). D. Cleavage sites of XerC^Vc and XerD^Vc on dif1 and dif2. Schemes of substrates are shown on the top of the gels. Suicide substrates were labeled on the 3′ side of the continuous strand, as indicated (3′*). PNK: phosphorylation with T4 polynucleotide kinase; G+A: chemical cleavage ladder. Sequences resulting from the chemical cleavage are indicated beside the gels. Bases of the central region and of the XerCD-binding sites are indicated in black and grey, respectively. The deduced cleavage points are indicated by black triangles.

https://doi.org/10.1371/journal.pgen.1000201.g003

Labeling the 5′-end of the continuous strand of suicide substrates allows the detection of covalent recombinase/DNA 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 3′ end. Cleavage by tyrosine recombinases generates a 5′OH DNA extremity whereas chemical cleavage leaves a 5′ 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⁻. 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).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 4. FtsK^Vc-dependent recombination at dif1 and dif2.

A. Reconstitution of V. cholerae Xer recombination at plasmid-borne dif1 and dif2 sites in E. coli cells. Top panel: gel showing a typical result. A scheme of the substrate and product bands is shown beside the gel. dif sites are represented by triangles. Bottom panel: quantification plot displaying the mean and standard deviations of at least three independent experiments. B. Recombination by wild-type (+) and catalytically inactive (YF) recombinases. HJ: HJ intermediate.

https://doi.org/10.1371/journal.pgen.1000201.g004

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). For both dif1 and dif2, no resolution product or HJ intermediate were detected in XerD_YF^Vc cells (Figure 4B, lane 2, 4, 6 and 8). In contrast, we could detect the accumulation of a HJ intermediate in XerC_YF^Vc XerD^Vc cells (Figure 4B, lane 3 and 7), 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.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 5. Species specificity in Xer recombination.

A. Amino acid residue conservation in the γ-domain of FtsK and in the C-terminal tail of XerD. Numbers indicate the position of the first and of the last residues of the alignments in the amino acid sequence of the V. cholerae proteins. Positions of full conservation and of strong or weaker groups of conservation are indicated by stars, semi-colons or dots, respectively, following the Clustal 1.83 scheme. Black bars and vertical arrows indicate residues implicated in FtsK-XerD interaction and in KOPS recognition in E. coli, respectively. B. Species-specificity in Xer recombination on plasmid-borne dif1, dif2 and dif^Ec sites. The mean and standard deviation of at least three independent experiments are plotted. ND: not determined. C. FtsK-independent recombination at dif^Ec by wild-type (+) and catalytically inactive (YF) V. cholerae recombinases. D. FtsK-independent recombination by the V. cholerae recombinases on hybrid dif sites.

https://doi.org/10.1371/journal.pgen.1000201.g005

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 γ-Proteobacteria consensus by only 2 bp, the substitution of A¹⁷ by G and the substitution of A¹⁰ 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). A site carrying the single [G-A]¹⁷ substitution promoted a much higher level of FtsK-independent recombination (dif12), while recombination at sites carrying the [T-A]¹⁰ and [G-A]¹ substitutions was not altered (dif13 and dif14). However, the cumulative substitutions of [G-A]¹⁷ and [T-A]¹⁰ increased FtsK-independent recombination to a level equivalent to dif^Ec-recombination (dif15). In addition, when T¹⁰ 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, G¹⁷ in the central region of dif1 and T¹⁰ in the XerC-binding site of dif1 and dif2 appear to have an important role in maintaining Xer recombination under the tight control of FtsK in V. cholerae.

V. cholerae FtsK Orienting Polar Sequences

We next investigated if FtsK^Vc could serve to bring together the CDR sites carried by dimers of chromosome I or by dimers of chromosome II. Several key residues implicated in KOPS recognition have been identified in the γ domain of FtsK^Ec (Figure 5A; N1296; R1300; E1303; [35]). The conservation of these residues in FtsK^Vc suggested that it could recognize the same motifs (Figure 5A; N926; R930; E933). If this was indeed the case, replacing the C-terminal domain of FtsK^Ec with the one of FtsK^Vc should completely rescue CDR in E. coli cells since FtsK^Vc fully activates recombination by XerCD^Ec at dif^Ec (Figure 5B, XerCD^Ec, dif^Ec, FtsK^Vc). Indeed, the fitness of such cells equaled the fitness of wild-type E. coli cells in growth competition experiments (Figure 6A, NLC^Vc and NLC^Ec), in contrast to cells only expressing the N-terminal domain of FtsK^Ec or a fusion with the C-terminal domain of H. influenzae FtsK (Figure 6A, N and NLC^Hi).

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 6. V. cholerae FtsK Orienting Polar Sequences.

A. Growth competition of E. coli cells encoding FtsK hybrids. N: cells carrying a complete deletion of the C-terminal domain and linker region of FtsK^Ec; NLC^Ec: cells carrying full length FtsK^Ec; NLC^Vc and NLC^Hi: cells in which the C-terminal domain FtsK^Ec was replaced by the one of FtsK^Vc and FtsK^Hi, respectively. f: frequency of cells that the parental N strain fails to produce at each generation compared to the FtsK hybrids. B. 5′-GGGCAGGG-3′ inhibits recombination activation by FtsK^Vc. Plasmid recombination at E. coli dif by XerCD^Ec was induced with 0.5% arabinose. Ec[NRE]: FtsK_50C^Ec[NRE]; Vc[NRE]: FtsK^Vc[NRE]. KOPS-0: substrate without GGGCAGGG sequences; KOPS-2: 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).

https://doi.org/10.1371/journal.pgen.1000201.g006

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 5′-GGGCAGGG-3′ 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 γ-Proteobacteria (Figure 7 and Figure S2). To determine dif sites in β- and α-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 γ-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 β-Proteobacteria species and 5 α-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 γ-, β- and α-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.

Download:

• PPT
  PowerPoint slide
• PNG
  larger image
• TIFF
  original image

Figure 7. The non-homologous chromosomes of Proteobacteria with multipartite genomes carry divergent dif sites.

Alignment of the chromosome dimer resolution sites of a few Proteobacteria harboring a single or multiple chromosomes. Bases identical to the γ-Proteobacteria dif consensus are shaded in black. Species abbreviations follow the KEGG convention.

https://doi.org/10.1371/journal.pgen.1000201.g007

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 β- and γ-Proteobacteria harboring a ‘canonical’ 5′-TGTATA-3′ 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, non-homologous chromosomes of bacteria with multipartite genomes, which competes with the selective pressure for dif central regions to adopt the preferential 5′-TGTATA-3′ 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 β2163 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 37°C with a 1000× dilution in fresh media every 12h [40]. Because of their higher growth rate, V. cholerae cells were grown at 30°C with a 10000× 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 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. 5′-end labeling of oligonucleotides was performed using T4 DNA polynucleotide kinase and [³²P] γ-ATP and 3′end labeling using terminal transferase and [³²P] α-ddATP. Reactions were performed in 20 mM Tris-HCl (pH 7.5), 50 mM NaCl, 0.1mM EDTA, 1 μg/ml of BSA, 40% glycerol and 0.2 pmol of radiolabeled probe for 2 hours at 37°C. 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 100× in LB and grown to 0.6 OD at 37°C. Cells were then grown for an extra 2 hours at 37°C 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.