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Sister chromatid interactions in bacteria revealed by a site-specific recombination assay

2012; Springer Nature; Volume: 31; Issue: 16 Linguagem: Inglês

10.1038/emboj.2012.194

1460-2075

Christian Lesterlin, Emmanuelle Gigant, Frédéric Boccard, Olivier Espéli,

Escherichia coli research studies

Article20 July 2012free access Sister chromatid interactions in bacteria revealed by a site-specific recombination assay Christian Lesterlin Christian Lesterlin Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, FrancePresent address: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK Search for more papers by this author Emmanuelle Gigant Emmanuelle Gigant Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, France Search for more papers by this author Frédéric Boccard Frédéric Boccard Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, France Search for more papers by this author Olivier Espéli Corresponding Author Olivier Espéli Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, France Search for more papers by this author Christian Lesterlin Christian Lesterlin Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, FrancePresent address: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK Search for more papers by this author Emmanuelle Gigant Emmanuelle Gigant Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, France Search for more papers by this author Frédéric Boccard Frédéric Boccard Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, France Search for more papers by this author Olivier Espéli Corresponding Author Olivier Espéli Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, France Search for more papers by this author Author Information Christian Lesterlin1,‡, Emmanuelle Gigant1, Frédéric Boccard1 and Olivier Espéli 1 1Centre de Génétique Moléculaire du CNRS, Gif-sur-Yvette, France ‡These authors contributed equally to this work *Corresponding author. Centre de Génétique Moléculaire du CNRS, Bât. 26, Avenue de la Terrasse, Gif-sur-Yvette 91198, France. Tel.:+33 1 69 82 32 14; Fax:+33 1 69 82 31 60; E-mail: [email protected] The EMBO Journal (2012)31:3468-3479https://doi.org/10.1038/emboj.2012.194 Present address: Department of Biochemistry, University of Oxford, Oxford OX1 3QU, UK PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The process of Sister Chromosome Cohesion (SCC), which holds together sister chromatids upon replication, is essential for chromosome segregation and DNA repair in eukaryotic cells. Although cohesion at the molecular level has never been described in E. coli, previous studies have reported that sister sequences remain co-localized for a period after their replication. Here, we have developed a new genetic recombination assay that probes the ability of newly replicated chromosome loci to interact physically. We show that Sister Chromatid Interaction (SCI) occurs exclusively within a limited time frame after replication. Importantly, we could differentiate sister cohesion and co-localization since factors such as MatP and MukB that reduced the co-localization of markers had no effect on molecular cohesion. The frequency of sister chromatid interactions were modulated by the activity of Topo-IV, revealing that DNA topology modulates cohesion at the molecular scale in bacteria. Introduction In all living cells, the inheritance of genetic material requires the faithful segregation of duplicated chromosomes into daughter cells. In eukaryotic cells, this process involves the alignment of chromatids upon replication, a process called sister chromatid cohesion (SCC) (Nasmyth and Haering, 2009). Newly replicated chromosomes remain tightly aligned and associated until the onset of mitosis. SCC is essential for genome stability since it is required for both high fidelity chromosome segregation and DNA damage repair. Furthermore, SCC is also involved in gene expression and cell development (Nasmyth and Haering, 2009). Cohesion is mediated by cohesin, a specific multi-subunit complex composed of "structural maintenance of chromosomes" (SMC) proteins. Other SMC complexes have a DNA condensing activity (Losada et al, 1998). The establishment of SCC on newly synthesized DNA is coupled with replication fork progression (Lengronne et al, 2006), and it is thought that cohesin topologically holds sister DNA strands together by trapping the DNA inside a molecular ring (Haering et al, 2002). The dissociation of cohesin complexes precedes a bipolar migration of sister chromosomes during mitosis. In prokaryotic cells, in contrast to eukaryotic cells, little is known about sister chromosome cohesion and its control. No molecular evidence revealing SCC at the molecular level has been reported, and no proteins with cohesin-like activity have been described. Bacteria contain SMC proteins that have been shown to play a role in chromosome organization and segregation in E. coli, C. crescentus and B. subtilis (Jensen and Shapiro, 1999; Danilova et al, 2007; Cui et al, 2008; Gruber and Errington, 2009; Sullivan et al, 2009). However, experimental evidences indicated a role in condensing-like rather than in cohesin-like activity (Petrushenko et al, 2006). SCC in bacteria has been proposed to occur based on cytological fluorescence imaging that revealed a colocalization period of newly duplicated loci, i.e., the period between the time a locus is replicated and the visual separation of the two sister loci. Depending on the growth conditions, the lifespan of loci colocalization varied (Adachi et al, 2008). Furthermore, sister chromatid co-localization is not constant along the E. coli chromosome. It was demonstrated that the region containing the migS site (200 kb from oriC) was segregated before the oriC locus while it is replicated later (Yamaichi and Niki, 2004). In the Ori macrodomain (MD), a large region of about 600 kb adjacent to oriC on the right replichore was more cohesive than the distal part of the replichore, the non-structured region (Bates and Kleckner, 2005; Espeli et al, 2008). An abrupt segregation of the region would trigger nucleoid splitting and segregation of the rest of the chromosome (Bates and Kleckner, 2005). Recently, it was shown that two particular regions inside the Ori MD, called snaps, present extensive colocalization as compared to the rest of the MD (Joshi et al, 2011). The Ter MD also displays significant sister co-localization (Espeli et al, 2008). A number of factors (MukB, MatP, Topo-IV) have been shown to modulate the period of sister loci colocalization (Sunako et al, 2001; Mercier et al, 2008; Wang et al, 2008). The effect of Topo-IV inactivation has led authors to propose that catenation might be a major contributor of SCC through colocalization of the sister loci (Wang et al, 2008). In the present work, we set-up a system that probes the ability of sister chromatids to interact physically, thus revealing sister chromatid cohesion directly in live cells. This new genetic system reports the probability of newly replicated copies of a locus colliding with each other. We used the extensively characterized site-specific recombination system of bacteriophage P1 Cre/loxP (Hamilton and Abremski, 1984) combined with a reporter gene to disclose sister chromatid molecular interactions (SCI) along the E. coli chromosome. The assay, called LacloxP, revealed that SCI is tightly controlled and lasts for 10–30 min following replication. Beside, the Laclox assay was used to test the involvement of several candidates in SCI. We showed that factors that promote co-localization of sister foci do not necessarily increase the ability of sister loci to recombine. Topo-IV was the only factor to display strong variation in sister chromatid interactions (SCI) when altered. The decatenation activity of Topo-IV was required to limit the extent of SCI, suggesting that precatenation links modulates post-replication cohesion between sister chromatids. Results The LacloxP assay to probe sister chromatid interactions We developed a genetic tool to reveal the physical interactions between sister chromatids in vivo (Figure 1A). This LacloxP assay was based on the Cre/loxP site-specific recombination system of the bacteriophage P1. The loxP cassettes were designed to detect recombination between sister chromatids. The lacZ gene was interrupted by two directly repeated loxP sites separated by 21 bp (lac2loxP in Figure 1A). β-galactosidase activity was retained in the presence of an in-frame fusion of a single loxP in lacZ (lac1loxP). The recombination of two loxP sites carried by the same molecule, i.e., an intramolecular/intrachromatid event (event 1 Figure 1A), was prevented by the proximity of the two loxP sites. The minimal distance required between two loxP sites for them to recombine was reported to be 82 bp; no recombination was detected in vitro below this distance (Hoess and Abremski, 1985). Thus, the reconstitution of an active lacZ gene that conferred the Lac+ phenotype (lac1loxP) was predicted to require recombination between the replicated sister LacloxP cassettes, i.e., an intermolecular/interchromatid event (event 2, Figure 1A). We introduced the LacloxP recombination cassette in six different chromosome loci: three close to the oriC (Ori-1, Ori-3 and Ori(SNAP)), three in the terminus region (Ter-1, Ter(dif) and Ter-6) (Figure 1B). To control recombination, the Cre recombinase gene was carried by a pSC101 plasmid and was under the control of the PBAD promoter. Induction of cre was achieved by the addition of 0.1% L-arabinose in order to ensure the homogenous induction of the PBAD promoter in the population. Figure 1.The LacloxP assay revealed sister chromatid interactions. (A) The LacloxP construct consisted of the lacZ gene (orange arrow) under a constitutive promoter that was interrupted by two directly repeated loxP sites (white arrows). This construct conferred the Lac− phenotype. The intramolecular recombination event (noted 1) was prevented by the proximity between the two loxP sites. After replication, intermolecular recombination occurred between sister chromatids (noted 2) when Cre was produced. This recombination forms the lac1loxP product (blue arrow), conferring the Lac+ phenotype. The intramolecular recombination between the first and third loxP sites of lac3loxP product is noted event 3. (B) Map of the E. coli chromosome with the position of the six loci tested in this study labeled. The positions are indicated in kb from the oriC. (C) Recombination events were revealed using PCR amplification that detected the substrate, lac2loxP (406 bp), and products, lac1loxP (351 bp) and lac3loxP (460 bp). The gel shows PCR amplification obtained on total DNA sample extracted from MG1655 strains containing the LacloxP system at the Ori-1 or Ter-6 loci before (NI) and after Cre induction for the indicated time. (D) LacloxP recombination happens on replicating chromosomes but not fully segregated chromosomes. Recombination events were revealed using PCR amplification following Cre induction in the MG1655dnaC2 strain. Replication initiation was blocked by a 2-h shift to a non-permissive temperature (40°C) simultaneously cytokinesis was prohibited by the addition of cephalexin (10 μg/ml). In these conditions the cells filamented and accumulated segregated nucleoids (data not shown). Cre induction was performed at 40°C for 20, 30 or 60 min, or following replication initiation after a downshift from 40 to 30°C for 20, 30 and 60 min. (E) LacloxP recombination follows the replication forks. We used the dnaC2 thermosensitive allele to synchronize the population. Cells were grown at 30°C until OD 0.2 and were shifted to 40°C for two hours to allow the completion of ongoing replications. Replication was initiated by a 10 min shift at 30°C, and cells were returned to 40°C to avoid over-initiation. Nine pulses of 10 min Cre induction were performed and then the cells were immediately harvested and frozen in liquid nitrogen. Genomic DNA was extracted and used for PCR amplification, revealing the formation of LacloxP recombination products. The quantification of recombination is presented (NRR), it corresponds to the measure of the amount of lac1loxP plus lac3loxP compared to the amount of lac2loxP. The values were normalized to that observed for the asynchronous culture. Download figure Download PowerPoint Recombination events between sister chromatid are rapidly detected by a PCR assay The formation of recombinant products, lac1loxP and lac3loxP, was detected by PCR assay (Figure 1C). The lac1loxP and lac3loxP products were detected 15 min after Cre induction and accumulated to high levels after 30 and 60 min, respectively; most of the lac2lox substrate was converted into lac1loxP and lac3loxP products. After 120 min, the reciprocal recombination product lac3loxP was hardly detected, indicating that it was converted into the lac1loxP product. In lac3loxP, the external loxP core sequences were separated by 105 bp and therefore supported intramolecular recombination in vivo (event 3, Figure 1A). Because of event 3 on the lac3loxP substrate, the amount of SC in the lac1lox configuration can nearly reach 100% upon a long cre induction. We first tested if lac2loxP recombination could occur between fully segregated chromosomes. We used an E. coli strain where replication initiation was under the control of a dnaC2, a thermosensitive allele of the DnaC initiation protein. At a non-permissive temperature (40°C), replication initiation was blocked, but ongoing rounds of replication were completed (Withers and Bernander, 1998). In the presence of cephalexin, which inhibits cytokinesis, the cells grew into filaments that harbored 4–8 fully replicated and segregated nucleoids. In these conditions, the induction of the Cre recombinase (for 20–60 min) did not result in the formation of any of the recombinant product (Figure 1D). This showed that, according to predictions, recombination did not occur between the directly repeated loxP sites that were spaced 21 bp apart on the same chromosome. Furthermore, recombination did not occur between fully segregated chromosomes. In contrast, when the temperature was shifted to 30°C concomitantly with cre induction, recombination products were observed after 20 min for the Ori-3 locus and after 60 min for the Ter-1 locus, consistent with their relative position on the replichores. This suggested that lac2loxP recombination was strictly dependant on replication of the locus. Sister chromatid cohesion during a limited time window after their replication We designed an experiment to determine the period of the replication cycle that is permissive for SCI (Figure 1E). To do so, we synchronized the cells using a thermosensitive allele of DnaC (dnaC2) that blocks initiation of replication at non-permissive temperature (40°C). The synchronization was achieved through three successive temperature shifts: (i) a 30–40°C shift for 2 h to terminate the ongoing round of replication; (ii) a 40–30°C shift for 6 min to initiate replication; (iii) a 30–40°C shift to inhibit further initiation of chromosome replication. We used flow cytometry to control the synchrony and measure the replication rate at 40°C (Supplementary Figure S1A); replication appeared to be completed in about than 40–50 min and half of the population had achieved cell division after 70 min (Supplementary Figure S1B). Assuming a linear progression of the forks, we approximated the times of replication of the Ori-1, Ori-3, Ter-1 and Ter-6 loci to be 5, 8, 35 and 36 min after initiation of replication, respectively.. The expression of cre was induced by 10 min pulses: one at 30°C before synchronization, two during the period of synchronization at 40°C (after 60 and 120 min), one during the temperature downshift at 30°C that triggered the initiation of replication, and finally five more during the subsequent incubation at 40°C. Cells were immediately frozen in liquid nitrogen after each pulse and genomic DNA was extracted. PCR amplification was performed to reveal the formation of lac1loxP recombination products (Figure 1E). Recombination was observed for every locus in the asynchronous cultures (pulse number 1). The amount of recombinant product lac1loxP in the asynchronous culture was used as a reference and normalized to 1 for the quantification of the recombination in the synchronized cells. The recombinant products were not detected after the inhibition of replication initiation at 40°C (pulses no 2 and no 3 in Figure 1E). Recombinant products were detected at early time points (10 and 20 min) following the synchronous replication initiation at the Ori-1 locus (no 4 and no 5 in Figure 1E) and were still detectable 30 and 40 min after replication initiation (no 6 and no 7). For the Ori-3 locus, recombinant products were detected 20 min after initiation of replication (pulse no 5) and were hardly visible 30 min after replication initiation (pulse No 6). For the Ter-1 andf Ter-6 loci, recombinant products were detected 40 and 50 mintes after replication initiation. These results indicated that recombination between sister chromatids can occur immediately after replication and only for a limited period of time, about 30 min for Ori-1 and 10–20 min for Ori-3, Ter-1 and Ter-6. Note that the amount of lac1loxP product detected by PCR was relatively low. This result suggested either that only a small fraction of the sister chromatids was maintained in a sufficient proximity to allow recombination or that the level of recombinant product was underestimated, perhaps because part of the long-lived Cre/loxP synapse intermediate was not a suitable template for PCR amplification (see below). Lac+ colonies formation is an accurate measure of SCI While the direct detection of recombinant products was enlightening the temporal control of SCI, because of the strong constraints imposed by the synchronization procedure, this approach was not appropriate to quantify SCI and analyze parameters that may modulate SCC. We used the same intermolecular recombination assay described above but the frequency of recombination (forming lac1loxP product) was measured according to the amount of Lac+ colonies observed on X-Gal plates (Figure 2). To assess the level of SSC in different regions of the chromosome, we performed the recombination assay with lac2loxP cassettes inserted at six different positions (Ori-1, Ori-3, Orisnap, Ter-1, Ter-6 and Terdif). Differential abilities of chromosome regions to promote LacloxP recombination could reflect either differential abilities for sister chromatids to interact or the difficulty for Cre to form synapsis between loxP sites in some regions of the chromosome. To discriminate between these two possibilities, we also measured for the same loci the capacity of intramolecular loxP/Cre recombination to estimate the relative access of Cre to different chromosomal regions (Figure 2A). The intramolecular recombination cassette contains two directly repeated loxP sites spaced by the rifampicin resistance gene: the 1 kb intervening segment separating the two loxP sites ensured efficient synapse formation in cis (Figure 2A). We recorded the number of recombinants as a function of Cre induction time for the six loci carrying cassettes for the intermolecular or the intramolecular assay (Figure 2B and Supplementary Figure S2C). The curves for the intramolecular recombination followed simple enzymatic rules: a short lag is observed (<1 min), then a robust and linear recombination rate is observed for every locus (∼10–13%/min). After 10 min of induction, a plateau is reached at approximately 100%. The curves for the intermolecular recombination differed slightly. We observed a longer lag (∼3 min) followed by a linear recombination rate (∼3–7%/min). A plateau (70–80% of Lac+ colonies) is reached after 25–40 min of induction (Figure 2B). Figure 2.Lac+colonies formation is an accurate measure of SCI. (A) Description of the inter and intramolecular recombinations. The intermolecular reaction, as described on Figure 1A, involves sister chromatid exchange between loxP sites (SCE). The intramolecular recombination between loxP sites distant from 1 kb can be monitored at every loci tested by the deletion of a Rif cassette and the reconstitution of the lacZ gene. It is used to control Cre accessibility at the different loci tested and the influence of the genetic background on Cre reactivity. Both inter and intramolecular recombination frequencies can be measured on plate by the formation of blue colonies. (B) Measurement of the frequency of SCI (inter-SC) and intramolecular recombination (intra-SC) according to the extent of Cre induction. The curves represent the average of four experiments. (C) Measure of the frequency of SCI by Lac+ colonies counting at the Ori-3 locus (inter SC). Following Cre inductions for 10–150 min after induction the cells were diluted 104 fold and immediately plated (dark gray). After the 10 min induction, cells were diluted 104 fold and kept in liquid culture with shaking for a delay up to 140 min before plating (light gray). (D) PCR analysis of the recombination products observed for the Ori-3 locus in the same induction conditions than on Figure 2C. Genomic DNA was extracted immediately after induction or after the indicated dilution delay. The amount of recombined products (3 loxP+1 loxP) was measured by density scanning with a Typhoon after electrophoresis (AU arbitrary densitometry unit). The arrow indicates the timing of the dilution. (E) Measure of the ability of Cre to produce recombined colonies following translation inhibition by the addition of chloramphenicol (Cm). The recombination frequency at the Ori-3 inter SC and Ori-3 intra SC cassette was measured by Lac+ colonies counting in the absence of Cm (gray boxes) or in the presence of Cm (120 μg/ml) 5 min after the addition of arabinose (blue boxes), the cultures were incubated at 30°C for the indicated times, then diluted 104 fold and immediately plated. Download figure Download PowerPoint In order to quantitatively relate the level of recombination to the molecular cohesion, we tested if the rate of recombination of the Ori-3 locus was dependent on the time left after induction and before plating. Following cre induction, cultures were diluted to stop the induction, and either immediately plated or kept at 30°C for 15–140 min before plating (Figure 2C). When the cultures were immediately diluted and plated, the frequency of Lac+ colonies observed on plate raised quickly to 100%. Strikingly, increasing the delays before plating did not change the frequency of Lac+ colonies. Similar results were observed with the the Ori-1 and Ter-1 loci (Supplementary Figure S2A). These observations suggest that most of the recombination took place during the induction pulse. The amount of recombined DNA was also directly monitored by PCR analysis (Figure 2D) or Southern blot (Supplementary Figure S2B); they revealed that recombined Ori-3 products were not efficiently detected immediately after the recombination pulse, they accumulated during the dilution step to reach a plateau (∼40% for a 10 min pulse, i.e. a similar amount than Lac+ colonies). These experiments indicated that recombination intermediates formed at the end of the induction period were converted into recombinant products during the 30 min that followed cre induction. These observations imply a rapid inactivation of the Cre recombinase following dilution of the arabinose inductor. We confirmed that the formation of Lac+ colonies did not rise after induction by using chloramphenicol to block translation at the end of the induction pulse (Figure 2E). The intramolecular recombination cassette was used at the Ori-3 position to measure recombination in the presence of chloramphenicol (for 5–35 min after induction). As observed for the dilution experiments, the amount of Lac+ colonies did not increase after inhibition of translation. These experiments indicated that the amount of Cre able to perform new recombination on SC formed in the minutes following the induction pulse is very limited. Therefore the number of recombinant resulted from the number of loxP synapses that formed during the induction time or shortly after; discrepancies between the PCR and the plating assays likely originated from a slow resolution of the synapse into recombination products. The amount of recombinant colonies on plate is likely the best quantification of the number of cells presenting molecular cohesion for the defined locus in the population for a given time interval, i.e. sister chromatids involved in molecular cohesion. LacloxP recombination reveals variability in sister chromatid cohesion along the chromosome Considering that intramolecular recombination is possible at any step of the E. coli cell cycle, we postulated that the intermolecular recombination rate relates to the fraction of the cell cycle when SC are available for recombination, i.e. the cohesion period. After the initial lag, both intermolecular and intramolecular reaction were following a single exponential decay (Amount of Lac- colonies)=Ae−kt. A is a constant that is close to 150% for the intermolecular reaction and close to 300% for the intramolecular reaction, t is the length of the induction period and k is catalytic constant of each reaction (sec−1) (Figure 3A and Supplementary Figure S2D). For the intramolecular reaction, k varies from 2 to 6.5 × 10−3 s−1 suggesting that Cre recombination is not equivalent in every region of the chromosome. These values are in the same range as that measured in vitro (3–7 min per event) confirming that Cre recombination is not a fast reaction (Fan, 2012). For the intermolecular reaction, k varies from 0.7 to 1.5 × 10−3 s−1 (Figure 3B). Considering that when the SC are in close contact the inter and intramolecular reactions are equally efficient, the ratio kinter/kintra gives an estimation of the portion of the cell cycle allowing inter SC recombination. We estimated that SC were in contact for 18 min (23% of the 80 min cell cycle) at the Ori-1 locus, 20 min at the Ori-3 (25% of 80 min) locus, 10 min (13% of 80 min) at the Ter-1 locus and 11 min (14% of 80 min) at the Ter-6 locus (Figure 3B). Since intramolecular recombination efficiency were comparable for the Ori and Ter loci, it was unlikely that the difference in the estimated cohesion would be caused by the higher level of recombination resulting from a higher copy number of the Ori loci compared to the Ter loci. We measured the kinetic parameter of SCI for two other loci known for an extended colocalization of newly replicated sister chromatid, in the snap region (Orisnap) (Joshi et al, 2011) and near the dif site (Terdif) (Espeli et al, 2008). SC at the Orisnap and Terdif loci, respectively, support intermolecular recombination for 32% of the cell cycle (26 min) and 40% of the cell cycle (32 min) (Figure 3B, Supplementary Figure S2B and C). SCI was high for the snap and dif regions; it suggested that the LacloxP assay and the colocalization of FROS tags were able to catch similar constraints on the SC. These observations suggested that the frequency of LacloxP recombination is an accurate tool to monitor SC proximity. Figure 3.Sister chromatid interaction varied according to the locus considered. (A) Cre recombination follows single exponential decay kinetics for the inter and the intramolecular recombination reactions. The data from Figure 2B were plotted according to the disappearing of Lac− colonies versus the length of the induction. Single exponential fits are represented. To avoid taking into consideration the lag observed during the first 5 min, the fits exclude the time 0 and 2.5 min of induction. (B) Kinetic constants, kinter and kintra were presented for every reaction. The estimation of the Cohesion Period was given by CP=τ × (kinter/kintra), where τ is the generation time (80 min). Download figure Download PowerPoint Distinction of SCI and colocalization of loci Sister loci colocalization has been extensively used as an indirect measure of the cohesion period in both eukaryotic (Straight et al, 1996) and bacterial cells (Sunako et al, 2001; Bates and Kleckner, 2005; Espeli et al, 2008). In bacteria, the FROS systems, the parS/ParB-GFP systems and the FISH technique have been used to monitor SC colocalization. However, these approaches remain unsatisfactory and controversial because colocalization varies according to the growth rate (Adachi et al, 2008), it does not necessarily imply that sister loci can interact physically and it relies on a difficult determination of the cell cycle parameters, C and D periods, in bacteria, (Nielsen et al, 2007; Adachi et al, 2008; Espeli et al, 2008; Lesterlin et al, 2008). The quantification of the number of SSB-Ypet foci and their repartition according to the cell size is a precise way to define the C and D period (Reyes-Lamothe et al, 2008; Espeli et al, 2012). The characterization of the cell cycle parameters according to the SSB-Ypet pattern (Figure 4A and B) is presented on Figure 4C. New-born cells were finishing the replication rounds initiated in the mother cell; they presented two SSB-YPet foci corresponding to the forks that replicated the right and left replichores. Later, the two foci merged at mid-cell for the replication of the terminus region. After a period without replication (cells lacking SSB-YPet focus), the replication was initiated simultaneously on two chromosomes (large cells with two SSB-YPet foci). A few minutes before division, the replication forks separated, giving rise to cells with 4 SSB-Ypet foci. The amount of cells without S