The Two Cis-Acting Sites, parS1 and oriC1, Contribute to the Longitudinal Organisation of Vibrio cholerae Chromosome I

Abstract

The segregation of bacterial chromosomes follows a precise choreography of spatial organisation. It is initiated by the bipolar migration of the sister copies of the replication origin (ori). Most bacterial chromosomes contain a partition system (Par) with parS sites in close proximity to ori that contribute to the active mobilisation of the ori region towards the old pole. This is thought to result in a longitudinal chromosomal arrangement within the cell. In this study, we followed the duplication frequency and the cellular position of 19 Vibrio cholerae genome loci as a function of cell length. The genome of V. cholerae is divided between two chromosomes, chromosome I and II, which both contain a Par system. The ori region of chromosome I (ori_I) is tethered to the old pole, whereas the ori region of chromosome II is found at midcell. Nevertheless, we found that both chromosomes adopted a longitudinal organisation. Chromosome I extended over the entire cell while chromosome II extended over the younger cell half. We further demonstrate that displacing parS sites away from the ori_I region rotates the bulk of chromosome I. The only exception was the region where replication terminates, which still localised to the septum. However, the longitudinal arrangement of chromosome I persisted in Par mutants and, as was reported earlier, the ori region still localised towards the old pole. Finally, we show that the Par-independent longitudinal organisation and ori_I polarity were perturbed by the introduction of a second origin. Taken together, these results suggest that the Par system is the major contributor to the longitudinal organisation of chromosome I but that the replication program also influences the arrangement of bacterial chromosomes.

Author summary

Proper chromosome organisation within the cell is crucial for cellular proliferation. However, the mechanisms driving bacterial chromosome segregation are still strongly debated, partly due to their redundancy. Two patterns of chromosomal organisation can be distinguished in bacteria: a transversal chromosomal arrangement, such as in E. coli, where the origin of replication (ori) is positioned at midcell and flanked by the two halves of the chromosome (replichores), and a longitudinal arrangement, such as in C. crescentus, where ori is recruited to the pole and the replichores extend side by side along the long axis of the cell. Here, we present the first detailed characterization of the arrangement of the genetic material in a multipartite genome bacterium. To this end, we visualised the position of 19 loci scattered along the two V. cholerae chromosomes. We demonstrate that the two chromosomes, which both harbour a Par system, are longitudinally organised. However, the smaller one only extended over the younger cell half. In addition, we found that disruption of the Par system of chromosome I released its origin from the pole but preserved its longitudinal arrangement. Finally, we show that the addition of an ectopic ori perturbed this arrangement, suggesting that the replication program contributes to chromosomal organisation.

Introduction

Bacterial chromosome replication is initiated from a unique origin (oriC) and progresses bidirectionally. The replication of circular chromosomes terminates opposite the oriC in a region termed the terminus (Ter). Within the Ter region is a site-specific recombination site, termed dif, dedicated to the resolution of chromosome dimers [1]. These factors define two replication arms, Left and Right, mirrored by the <oriC-dif> axis. Detailed investigations of the choreography of chromosomal movements during the cell cycle of several monochromosomic bacteria suggest that segregation is concurrent with replication and starts with the precise positioning of newly replicated sister copies of the oriC region into opposite cell halves [2], [3], [4], [5]. Segregation of other sister chromosomal loci to their positions in daughter cells follows shortly after their replication with sister copies of Ter being segregated last [2], [3], [4], [5]. Less is known about the choreography of chromosome segregation in bacteria with multipartite genomes. However, the analysis of a single locus in the oriC region and a single locus in the putative Ter region of the two Vibrio cholerae chromosomes suggests a model of replication and segregation that is consistent with monochromosomic bacteria [6], [7], [8]. Taken together, these observations suggest that the active positioning of the oriC region sets the pace for chromosome segregation, raising questions regarding the underlying mechanism.

Similar to most other bacteria, a specific partition system is encoded on each of the Vibrio cholerae chromosomes [9]. Bacterial chromosome partition machineries are related to the Type I partitioning systems of plasmids. They consist of two genes, parA, which codes for an ATPase, and parB, which codes for a sequence-specific DNA binding protein that is able to spread around its binding site, parS [10]. Several parS sites are usually found proximal to and in some cases encompassing the oriC region of bacterial chromosomes [9]. The role of Par systems in DNA segregation is well established for low-copy number plasmids [11], [12], [13]. However, their role in bacterial chromosome segregation remains controversial, notably because the disruption of Par systems in different bacterial species produces different phenotypes. The Par systems of Caulobacter crescentus, Myxococcus xanthus and V. cholerae chromosome II are essential for chromosome segregation [4], [14], [15]. The impairment of the Pseudomonas aeruginosa Par system caused the formation of ∼20% anucleate cells [16]. However, the disruption of the Par machinery affects few cells (<2%) in Bacillus subtilis [17] and yields no segregation defect for V. cholerae chromosome I [18]. Moreover, several bacterial species, notably Escherichia coli and Streptococcus pneumonia, lack a functional Par system. Partition systems have also been implicated in other cellular processes including replication initiation [19], [20], cell cycle coordination [4], [14] and chromosome compaction [21], [22], [23]. Finally, ParB-binding to oriC-proximal parS sites recruits SMC proteins to the origin region in B. subtilis and S. pneumoniae, contributing to chromosome segregation even in the absence of ParA [21], [22], [23]. Nevertheless, Par systems are directly involved in the polar positioning and the active bipolar migration of the oriC region of the C. crescentus chromosome and V. cholerae chromosome I [8], [24], [25], [26], [27]. The polar anchoring mechanisms have been described for these two systems [27], [28].

The characterization of chromosomal organisation in different bacterial species suggests a common mechanism of longitudinal organisation in which the oriC region is positioned towards the old pole, Ter is positioned towards the new pole, and the two chromosome replichores extend over the long axis of the cell [3], [4], [29], [30]. The polar localisation of the oriC regions of V. cholerae chromosome I and of the multiple chromosomes of S. meliloti and A. tumefaciens suggests a similar longitudinal organisation [31], [32]. In contrast, the E. coli chromosome, which seems devoid of a Par system, adopts a transversal organisation where the oriC region is positioned at midcell and the left and right chromosomal arms extended toward the opposite cell halves [33], [34]. Taken together, these observations suggest that it is the active positioning of the oriC region towards the old cell pole by Par systems that is responsible for the longitudinal chromosomal organisation observed in most bacterial species.

However, two characteristics of V. cholerae make it an ideal bacterial model to test this hypothesis. Firstly, the Par system of V. cholerae chromosome II positions the origin region (oriC2) at midcell rather than at the cell pole, which would be expected to drive a transversal arrangement if the hypothesis was correct. Secondly, disruption of the Par system of V. cholerae chromosome I does not affect any step of the cell cycle, allowing for the direct investigation of its contribution to chromosomal organisation. In this current study, the analysis of the intracellular location of 12 chromosome I loci and 7 chromosome II loci in exponentially growing cells indicated that both V. cholerae chromosomes adopted a longitudinal organisation. Chromosome I extends from the old pole to the new pole and chromosome II extends from midcell to the new pole, i.e. in the younger cell half. By displacing parS1 sites away from oriC the Par system was shown to contribute to the longitudinal organisation of chromosome I. However, the parS1-deleted chromosome I remained longitudinal, with the oriC locus remaining located close to the old pole. The insertion of an ectopic origin of replication was sufficient to disrupt the longitudinal organisation of parS1 deleted chromosome I. Interestingly, the ectopic origin of replication was often positioned closer to the old pole than the original oriCI. Taken together, these observations suggest that the replication program contributes to the longitudinal organisation of V. cholerae chromosome I.

Results

Fluorescent labelling systems and analysis of chromosome choreographies

Chromosome choreography involves successive steps of chromosome organisation as a function of cell cycle progression. To avoid any complications linked to multiple concurrent rounds of replication, cells were cultivated at 37°C in slow growing conditions (M9 Fructose supplemented with thiamine) characterised by a 55 min generation time divided into three successive periods: a period of 11 min before replication initiation (B period), a 32 min-long replication period (C period) and a 12 min period after replication and before division (D period) [35]. Snapshot images of cells in steady state exponential growth were acquired. By correlating the length of cells with their progression through the cell cycle we studied chromosomal organisation as a function of cell elongation. Cells were classified according to their length in 0.1 µm intervals. A sliding window of 0.3 µm was used to determine the median position and frequency of duplication of any specific chromosomal locus. In our growth conditions the majority of cells had a length between 1.9 µm and 4.3 µm. We will refer to the cells of the first point plotted, corresponding to the 2 µm interval, as newborn cells and the cells of the last interval plotted, corresponding to the 4.2 µm interval, as dividing cells.

The spatial organisation of chromosome I and of chromosome II was deduced from the positioning of 12 and 7 loci respectively. They were broadly distributed over the entire genome, particularly the regions of special interest, such as the origin of replication and the chromosomal dimer resolution site of each of the two chromosomes (ori_I, ori_II, ter_I and ter_II). Five loci were tagged on the right (R1_I to R5_I) and left (L1_I to L5_I) replichores of chromosome I. On chromosome II, three loci were tagged on the right replichore (R1_II to R3_II) and two on the left (L1_II and L2_II). The loci were visualised in pairwise combinations using two compatible fluorescent labelling systems, a lacO array was inserted at one of the loci and a parS _pMT1 site at the other locus. LacI-mCherry and yGFP-Δ30ParB_pMT1 [34] protein fusions were produced from an operon in place of the V. cholerae lacZ gene. In some cases, a third locus was tagged with a tetO array and visualized by the production of a TetR-Cerulean fusion from a plasmid. We observed the same pattern of localisation of the L3_I or terI loci during the cell cycle whether they were tagged with a lacO or tetO array or with a parS _pMT1 site, suggesting that these three systems did not affect the positioning of the chromosomal loci under our experimental conditions.

Dual labelling has three advantages. Firstly, the precise orientation of the long axis of the cell from the new pole to the old pole could be determined. The new pole is defined as the pole resulting from the previous cell division event. Assuming no gross rearrangements of chromosomal DNA after cell division, a locus closer to the septum than to the poles in dividing cells was defined as being closer to the new pole than to the old pole in newborn cells. Time-lapse microscopy observations validated this method (Figure S1). Secondly, dual labelling allowed for the collection of data in two different strains even if they presented slightly different cell size distributions (Figure S2). The cell size distribution of the two strains was re-aligned using the frequency of duplication of a common locus as a reference. In the rest of the manuscript we will refer primarily to compiled figures. However, each of our conclusions could be drawn based on individual strain data. The data of each individual strain are presented in Figures S3 to S39. Thirdly, dual labelling allowed for the comparison of the timing of duplication of the two tagged loci of each strain as a function of cell elongation and the direct measure of the distance separating these loci as a function of cell elongation. These comparisons could be made independently of any orientation procedure or cell size re-alignment.

Sequential order of duplication of chromosomal DNA in V. cholerae

The tagged loci were always observed as a single focus in newborn cells (Figure 1B and 2B). This is consistent with our growth conditions in which newborn cells contain a unique non-replicating copy of each of the two chromosomes. In dividing cells, two separate foci were observed for most of the analysed loci (Figures 1B and 2B). The only exceptions were L5_I, R5_I and ter_I, whose foci were duplicated in about 50%, 40% and 10% of the dividing cells, respectively (Figure 1B). This is consistent with a previous report where sister copies of a locus situated at 40 kbp from dif1 remained colocalised until the very end of septation [6].

Figure 1. Longitudinal organisation of V. cholerae chromosome I: Sequential duplication and segregation.

(A) Circular V. cholerae chromosome I and II maps indicating the position of the different tags with respect to oriC1, parS1 and matS sites and their corresponding colour code. (B) Proportion of 2 foci cells according to the realigned cell length (cell size intervals of 0.1 µm) for the different loci of the chromosome I. For each locus, a minimum number of 800 cells were analysed. Left panel: left replichore; Right panel: right replichore. The first cell size interval where ≥50% of cells contained two L3_I foci served to align the cell length distributions of ADV20, ADV21, ADV22, ADV23, ADV25, ADV33, ADV42, ADV50 and ADV51 strains, using ADV24 L3_I as reference. The strain EPV213 was aligned against ADV42 using the timing of recruitment of ter _I to midcell. (C) Reconstitution of the segregation choreographies of the 12 chromosome I loci. Left panel: left replichore; Right panel: right replichore. The median, the 25^th and the 75^th percentiles of the relative cell position of each locus are plotted for each cell size interval. The cells falling into the first interval were named newborn cells and the ones falling into the last interval were named dividing cells. 0: new pole; 1: old pole. (D) The relative distance between different chromosome I loci to the L3I locus was measured as a function of the relative cell length in the cells containing only one focus of each locus. The median (horizontal bar), the 25th and the 75th percentiles (open box) and the 5th and the 95th percentiles (error bars) of the distance of a given locus to L3I were indicated at this locus position along a chromosome I linear genetic map. (E) Relative distance between any of the chromosome sister loci, measured in cells with a length >3.4 µm.

Figure 2. Longitudinal organisation of V. cholerae chromosome II: Sequential duplication and segregation.

(A) Circular V. cholerae chromosome II map indicating the position of the different tags with respect to oriC2, parS2 and matS sites and their corresponding colour code. (B) Proportion of 2 foci cells according to the realigned cell length (cell size intervals of 0.1 µm) for the different loci of chromosome II. For each locus, a minimum number of 800 cells were analysed. The first cell size interval where ≥50% of cells contained duplicated L3_I was used to realigned ADV26 to the ADV24 reference strain. CP708, ADV131, ADV30 and ADV131 cell size distributions were aligned with the GDV552 cell sizes based on the cell size interval where ≥25% of cells contained two ter_II loci. GDV552 was aligned to ADV42 using the cell size interval where ter_I is recruited to midcell. ADV123 was aligned with ADV24 using the first cell size interval where ≥50% of cells contained two ori_I. (C) Reconstitution of the segregation choreographies of the 7 chromosome II loci, as in Figure 1C. (D) Relative distance between any of the chromosome II loci to the ter_II locus, measured in the cells containing only one focus of each locus. (E) Relative distance between any of the chromosome sister loci, as in Figure 1E.

The order of duplication of sister copies of each locus as a function of cell elongation followed the genetic map from ori to ter along the two arms of chromosome I and of chromosome II (Figures 1B and 2B). In addition, with the exception of the dif1 proximal loci, the rate of focus duplication of any given locus was similar. The proportion of cells with two foci increased abruptly from 10% to above 80% within ≤0.6 µm of cell elongation. However, the difference in the timing of duplication of two consecutive loci was not strictly proportional to the genetic distance that separated them. For instance, sister L1_I separation immediately followed sister ori_I separation and occurred much earlier than sister L2_I separation, despite L1_I being closer to L2_I than to ori_I. This observation is consistent with a previous finding in E. coli suggesting that the time sister loci remain together after replication is variable [36]. In addition, it was sometimes not possible with the resolution of our experiments to differentiate the timing of duplication of loci that were too close on the genetic map, such as ori_II, R1_II and R2_II or R3_I and R4_I.

Finally, using a strain with tagged ori_II and L3_I, it was observed that the separation of sister L3_I occurred at smaller cell length than for sister ori_II (Figure S9, ADV26). These results suggest that the separation of sister copies of the loci most proximal to the origin of replication on chromosome II occurred later than sister ori_I separation (Figures 1B and 2B). This observation is in agreement with the delayed firing of the origin of replication of chromosome II compared to chromosome I replication [35].

Longitudinal organisation of Chromosome I and II

For most loci and cell length categories, we observed cells with either two separated foci or a single focus. The proportion between these two types of cells varied as a function of cell elongation (Figures 1B and 2B). To simplify the representation of the data, we plotted the position of the foci that correspond to the dominant cell type in each cell length interval (Figures 1C and 2C). In addition, we plotted the median positions of the observed foci (filled circles), along with the 25^th–75^th percentiles (error bars).

In newborn cells, the relative cell position of ori_I and ter_I was approximately 0.8 and 0.1 respectively, reflecting that ori_I is positioned near the old pole and ter_I close to the new pole. In agreement with previous reports the position of ori_I in the overall cell population, i.e. irrespective of cell length, was closer to 0.9 [8], [20]. The distance between the position of ori_I and the position of any locus on chromosome I correlated with the genetic distance, suggesting a longitudinal arrangement of the replichores within the cell (Figure 1C). To confirm this longitudinal arrangement we directly calculated the distance between L3_I, which is located approximately in the middle of the left replichore, to additional chromosome I loci in cells that displayed a single focus for each of these two loci (Figure 1D). Consistent with a longitudinal arrangement L3_I was observed as being closer to the right replichore loci than to ori_I and ter_I (Figure 1D). Furthermore, we directly calculated the distance between separated sister loci in individual cells (Figure 1E). The distance between sister copies of each locus linearly decreased from ori_I to ter_I, further confirming the longitudinal organisation of chromosome I (Figure 1E).

Two modes of segregation could be distinguished among chromosome I loci. The first mode of segregation was observed for loci located from ori_I to L4_I or R4_I. In this mode, loci remained close to the position they occupied at cell birth until their duplication. As duplication occurred the greater the genetic distance between a locus and ori_I, the longer it remained static at its home position. After duplication sister foci segregated to their new home positions in the next cell length interval, suggesting that segregation might be a transient event within the cell cycle. The distances travelled by two sister foci were not identical. The most unbalanced situation was observed for ori_I where one copy remained nearly immobile whilst the second copy crossed the whole cell length. In contrast, the two L3_I sister loci exhibited a symmetrical separation on either side of the midcell/future new pole. The second mode of segregation was characterized by the mobilization of the loci towards midcell before duplication. This mode of segregation applied to loci located in the terminus region of chromosome I, i.e. R5_I, L5_I and ter_I. These loci migrated towards midcell (within 4 intervals) earlier than the duplication of loci located several hundreds of kb upstream (Figure 1B).

The ori_II of chromosome II positioned near midcell (with a relative position of 0.55) and dif2 near to the new pole (with a relative position of 0.28) in newborn cells. This is consistent with previous reports [6], [8]. The positioning and mobilisation of the ori_II region depends on a partition system like ori_I [15]. The positions of R1_II, R2_II, R3_II and L1_II, L2_II were intermediate between those of ori_II and ter_II, suggesting that chromosome II occupied only the younger half of the cell. Despite the short distance separating ori_II proximal loci (Figure 2B), the sequential ori _II to ter _II positioning of the seven chromosome II loci was observed, suggesting a longitudinal organisation (Figure 2C). Direct measurement of the distance between ter _II and R1_I, R2I, R3_I and L2_II (Figure 2D) and of the distance between sister copies of all of the chromosomal loci (Figure 2E) confirmed the longitudinal organisation of chromosome II. Furthermore, the duplication of most loci occurred close to midcell (Figure 2C). This suggests that un-replicated chromosome II loci are pulled towards midcell by replication, which in turn pushes sister copies of replicated loci away from midcell and each other.

Impact of parS1 displacement on chromosome I organisation

We speculated that the displacement of parS1 away from oriCI would modify the longitudinal arrangement of chromosome I. To test this hypothesis, tandem parS1 sites were introduced at 300 kb, 490 kb and 650 kb on the left arm of chromosome I in cells devoid of their three natural parS1 sites. Displacing the location of the Par-mediated anchoring zone on chromosome I did not affect the fitness or the morphology of the cells. The functionality of the displaced parS1 sites was not affected as judged by the visualisation of at least one polar focus of Ypet-ParB1 in the majority of different cell populations (Figure S40).

To assess the effect of parS1 displacements on the global organization of chromosome I, the cellular positions of ori_I, R2_I, L3_I and ter_I were visualized. Displacing parS1 sites on chromosome I had a dramatic effect on the positioning and segregation of ori_I, L3_I and R2_I (Figure 3B). In contrast, the positioning and segregation of ter_I was not significantly affected (Figure 3B). However, the displacement of parS1 to the L3_I location affected the polar positioning of ter_I prior to its final recruitment to midcell.

Figure 3. Reorganisation of chromosome I upon parS1 displacement.

(A) Circular map indicating the position of the different loci analysed in different genetic background (WT, parS1 _{300 kb} and parS1 _{650 kb} from left to right, respectively) and the colour code of the analysed loci. (B) Reconstitution of the segregation choreographies, as in Figure 1C. 2 µm of cell elongation were shown centred on the cell interval where at least 50% of the cells had duplicated ori_I. For the WT choreography, ADV114 and ADV78 cell size distributions were aligned with the ADV24 cell sizes using the first cell size interval where 50% or more of cells contained duplicated ori_I. For the parS1 _{300 kb} choreography, CP604 and CP582 cell size distributions were aligned with the CP591 cell sizes using the first cell size interval where ≥50% of cells contained 2 foci of ori_I and L3_I, respectively. For the parS1 _{650 kb} choreography, the cell sizes correspond to strain CP605 (ori_I and ter _I). CP583 and CP586 cell size distributions could not be realigned. The corresponding data was plotted with dashed lines (L2_I and R2I). (C) Relative distances between any of the sister loci, as in Figure 1E. (D) Relative distance between any loci to ter_I locus, as in Figure 1D. (E) Relative distance between ori_I sister loci, in with displaced parS1 sites, as in Figure 1E.

The displacement of parS1 sites to a location equidistant to ori_I and L3_I, caused the single ori_I focus of small cells to shift towards midcell and the ori_I sisters to be positioned closer to the quarter positions in long cells (Figure 3B, parS1@300kbp). This was confirmed by a decrease in the distance between ori_I and ter_I before ori_I duplication (Figure 3D) and a decrease in the distance between ori_I sisters after their separation (Figure 3C and 3E). The single L3_I focus of small cells was shifted towards the old pole and L3_I sisters were positioned further away from quarter positions in long cells (Figure 3B, parS1@300kbp). This was confirmed by the direct measurement of the distance between L3_I sisters after their separation (Figure 3C). In addition, R2_I positioning was shifted toward the new pole, as observed by a reduced distance between sister R2_I loci (Figure 3C). As a consequence, L3_I and R2_I no longer co-localised (Figure 3B).

When the parS1 sites were displaced to the L3_I locus, the home position of ori_I and L2_I were switched. The ori_I locus, which was now situated 700 kb away from the parS1 sites, adopted the positioning of L3_I locus in the wild-type context (Figure 3B, parS1@650kbp). This was confirmed by a decrease in the distance between ori_I and ter_I before ori_I duplication (Figure 3D) and a decrease in the distance between ori_I sisters after their separation (Figures 3C and 3E). Reciprocally, L2_I, which was now located 170 kb from the parS1 sites, exhibited a positioning similar to ori_I in the wild-type cell (Figure 3B, parS1@650kbp). The distance between sister L2_I also became larger than the ori_I sister distance (Figure 3C). Finally, the shift of R2_I positioning toward the new pole was exacerbated (Figure 3B and 3C).

To further demonstrate the extent of the chromosomal reorganisations, we took advantage of our double labelling systems to directly monitor the respective positions of ori_I and L3_I within long cells once they were duplicated and had reached their new home position. We determined the proportion of cells in which ori_I was more polar than L3_I when the parS1 sites were at their normal position (parS1 _{65 kb}, wild-type) or had been displaced by 300 kb (parS1 _{300 kb}) or 490 kb (parS1 _{490 kb}). This method allowed for chromosome rearrangements to be monitored at the single cell level. ori_I was more polar than L3_I in almost 100% of the cells in the wild type context (Figure 4A, parS1 _{65 kb}). The proportion of cells in which ori_I was more polar than L3_I decreased to 60% when parS1 sites were equidistant between ori_I and L3_I (Figure 4A, parS1 _{300 kb}) and down to 30% when parS1 sites were closer to L3_I than to ori_I (Figure 4A, parS1 _{490 kb}). The median distances between separated ori_I sisters were very similar when parS1 sites had been displaced to 300 kb or 490 kb (Figure 3E).

Figure 4. Increase of L3_I polarity over ori_I upon parS1 or oriC1 proximity to L3_I locus.

For each pole of dividing cells, the most polar locus between ori_I and L3_I was determined (A) in WT (ADV24), parS1 _{300 kb} (CP591) and parS1 _{490 kb} (CP634) and (B) in ΔparS1 (CP568), ΔparS1 + oriC1 _{651 kb} (CP626), parS1 _{65 kb} + oriC1 _{651 kb} (CP633). This corresponds to approximately 100 cells. The red part of the stacked histogram represents the proportion of case with ori_I more polar than L3_I, whereas the green part represents the opposite.

In conclusion, the displacement of parS1 sites along the left arm of chromosome I led to the global rearrangement of chromosome I within the cell. These results suggest that the Par system of chromosome I not only mobilises and anchors the parS1 sites at the poles but also directly contributes to the arrangement of the entire DNA molecule. Only the positioning of the terminus region escaped the influence of the Par system.

Impact of parS1 deletion on chromosome I organisation

Previous studies indicated that deletion of parA1 resulted in the release of the parS1 sites from the old pole with the relative position of ParB1 changing from 0.023 in a wild-type context to 0.2 [6], [8]. Despite this dramatic change, no effects on cell fitness or cell cycle parameters were reported, suggesting that chromosome I segregation was not affected by the disruption of its Par system. However, we speculated that the organisation and segregation choreography of chromosome I would be globally modified when Par-mediated mobilization and anchoring of ori_I were lost. To this end, we followed the localisation of ori_I, R2_I, L3_I and ter_I loci in ΔparS1 cells. The longitudinal arrangement of chromosome I was maintained during the whole cell cycle. ori_I remained the closest loci to the old pole with a relative position of 0.7 in small cells and a relative ori_I - ter_I distance of 0.68 (Figures 5B and 5C). R2_I and L3_I loci remained co-localised during the whole cell cycle and were positioned around midcell until their duplication (Figure 5B). After duplication, ori_I sisters remained more polar than L3_I and R2_I sisters, which still co-localised (Figure 5B). However, the relative distance between separated ori_I sisters was smaller than in wild-type cells (Figure 5D). In addition, the increase in the 25^th–75^th percentiles (error bars, Figure 5B) and the increase in the variability of the ori_I - ter_I distance (Figure 5C) suggest that the home positions of ori_I, L3_I and R2_I before and after duplication were less stringently controlled. Time-lapse experiments confirmed the global behaviour of these three loci (data not shown). In contrast, the choreography and late duplication of ter_I was not affected by the absence of the parS1 sites. The positioning of ori_II, R2_II and ter_II loci suggest that the chromosome II behaviour was not dramatically modified in ΔparS1 cells (Figure S41). In conclusion, the impairment of the Par system of chromosome I slightly disorganized the positioning and the segregation of the bulk of chromosome I. However, chromosome I remained longitudinally organised and ori_I remained more polar than the rest of chromosome I, suggesting the existence of additional chromosome I organizing and positioning mechanisms.

Figure 5. Disorganisation of chromosome I upon the ectopic oriC1_{651 kb} addition.

(A) Circular maps indicating the position of the different loci analysed in different genetic background from left to right, respectively : ΔparS1, ΔparS1 + oriC1 _{651 kb}, parS1 _{65 kb} + oriC1 _{651 kb}. (B) Reconstitution of the segregation choreographies of the loci tagged in the different genetic background, as in Figure 1C. For the ΔparS1 choreography, the CP639 cell size distribution was aligned with the CP655 cell sizes using the cell size interval where ≥50% of cells contained two R2_I foci; For the ΔparS1 + oriC1 _{651 kb} choreography, the cell sizes of CP626 and CP656 were aligned to the cell sizes of CP659 using the cell size interval where ≥50% of cells contained two ori_I foci; For parS1 _{65 kb} + oriC1 _{651 kb} choreography, the cell sizes of ADV115 were realigned to the cell sizes of CP633 using the cell size interval where ≥50% of cells contained two ori_I foci. (C) Relative distance between ori_I and ter_I loci, as in Figure 1D. (D) Relative distance between the ori_I sister loci, as in Figure 1E.

Impact of an extra oriC1 at 651 kb on chromosome I organisation

We hypothesised that the replication program might be, at least in part, responsible for the maintenance of the longitudinal arrangement of chromosome I in the absence of parS1 sites. To test this hypothesis, we introduced an extra origin of replication 651 kb from its normal position on the left replichore of chromosome I in cells lacking parS1 sites. The fitness of cells harbouring the two origins was not affected. Replication profiling demonstrated that the ectopic origin was as efficient as the wild-type origin and that the two origins were used at each cell cycle (Figure S42). Correspondingly, the separation of ori_I and L3_I sisters became synchronous (Figure S42). The perturbation of the replication program further decreased the polarity of the ori_I region in small cells (Figure 5B), which decreased the distance between single ori_I and ter_I foci (Figure 5C). The effect was more evident in long cells (Figure 5B) where the relative distance between separated ori_I sisters decreased to half the distance of wild-type cells with a high level of variability (Figure 5D). The segregation of ori_I sisters also became asymmetric with one of the two copies remaining relatively polar while the other adopted a more central position (Figure 5D). Finally, the L3_I locus became slightly more polar and no longer co-localised with R2_I (Figure 5B). In contrast, earlier replication of the ter_I locus (Figure S42) did not modify its home position (Figure 5C). The position of the R2_I locus was not significantly affected by the ectopic origin (Figure 5C). Then, we decided to directly monitor in each cells which of the two competing locus, ori_I and L3_I, was closer to the old pole. If the polar location of a given locus was only dictated by its proximity to the initiation of replication the proportion of cells in which L3_I was closer to the old pole than ori_I would reach 50%. Correspondingly, the polarity of ori_I compared to L3_I decreased from 80% in the parental cells (ΔparS1) to 55% in the cells harbouring an ectopic origin on the Left arm at 651 kb from ori_I (Figure 4B). The polar location of ori_I compared to R2_I was unchanged by the addition of the ectopic origin, suggesting that the phenotype was not linked to a global disorganisation of the DNA within the cell (Figure 4B). Thus, the replication program itself contributes to the polar positioning of loci that are close to the origin of replication. However, this contribution is masked by the presence of a Par system (Figure 5B). The contribution of the replication program may be more evident in cells containing a unique but displaced oriC1 site. However, the presence of an oriC1 site within the origin region is essential for cell viability (Figure S43).

Discussion

In this manuscript the use of fluorescent microscopy to follow the position of 19 loci within the genome of V. cholerae provides the first detailed characterization of the organization and dynamics of a multipartite bacterial genome.

The two V. cholerae chromosomes are longitudinally arranged

After division, the two replichores of chromosome I were arranged side by side from the old pole to the new pole (Figures 1 and 6A, WT). This longitudinal organization, with the origin positioned near the old pole and the terminus near the new pole in newborn cells, was expected for chromosome I due to the similarities in the positioning and dynamics of the origin and terminus regions with C. crescentus [30]. Previous reports suggested that the origin region of V. cholerae chromosome II was positioned at midcell in newborn cells and that, after replication, sister copies of it migrated to ¼ and ¾ positions [8]. This is similar to the positioning and dynamics of the E. coli chromosome, which suggested a transversal mode of organization for chromosome II. However, we demonstrate that the two replichores of chromosome II were arranged side by side with chromosome II only occupying the new half of the younger cells and the two sister chromatids the central part of the older cells (Figures 2 and 6B, WT). Thus, both V. cholerae chromosomes are longitudinally organized within the cell.

Figure 6. Models of chromosome I and II organisation and reorganisations by parS1 and oriC1 actions.

(A) Chromosome I was divided in 4 regions Ori in green corresponding to the region proximal of the replication origin , Left in blue, Right in Red corresponding to the left and right replichores and Ter in Yellow corresponding to the matS sites containing region (circular map). In the WT context, the Ori and the Ter are confined to the old pole and new pole, respectively. The Left and Right are laying in between. In the ΔparS1 context, the longitudinal organisation is maintained but the Ori is detached from the pole. The loss of parS1 sites do not convert the organisation of chromosome I to a transversal type as in E. coli (crossed drawing). In the parS1 _{650 kb} context, the Left becomes more polar than the Ori region. The Right is restricted toward the new pole where the Ter remains positioned. In the oriC1 _{651 kb} context, the Left becomes as polar as the Ori but the chromosome is globally less organised. (B) The chromosome II was divided in 4 regions as for chromosome I. In the WT context, the Ori was confined to midcell. The Left and Right are extended from midcell to the new pole. The TerII is not closely tethered to the new pole as terI.

Positioning and segregation dynamics of the terminus region

The terminus region of chromosome I behaved differently from the bulk of the chromosome and was recruited to midcell long before the time of sister foci duplication (Figure 1). In addition, terminus sister copies remained together until the very end of the cell cycle (Figure 1). Finally, whereas displacing parS sites led to the rotation of the whole chromosome in C. crescentus [30], the position of the terminus region of chromosome I was not affected by parS1 displacements (Figure 3). These observations are likely due to the presence of a MatP/matS system in V. cholerae that is absent from C. crescentus [37], [38]. Our results also suggest that sister copies of the terminus region of chromosome II separated earlier than sister copies of the terminus region of chromosome I even though both regions harbour matS sites (Figure 2). The differential contribution of the MatP/matS macrodomain organization system to the segregation of the terminus regions of chromosome I and II is the subject of a another study [39].

Longitudinal organisation is not entirely driven by partition systems

As for all other studied bacterial chromosomes with a longitudinal organization the position of the origin of replication region of each of the two V. cholerae chromosomes was driven by a ParABS partition system [40]. We speculate that the longitudinal organization of each chromosome is linked to the action of their partition machineries. Consistent with this, the displacement of parS1 sites rearranged the bulk of chromosome I within the cell with the locus most proximal to the displaced parS1 sites now occupying the polar edge of the chromosome (Figures 3, 4A and 6A, parS1@650kb). This is similar to the impact of parS sites on chromosome organisation in C. crescentus [30]. However, chromosome I remained longitudinally arranged in the absence of parS1 sites suggesting the existence of additional factors involved in chromosomal organisation (Figures 5 and 6A, ΔparS1).

Replication contributes to chromosome segregation in V. cholerae

In C. crescentus sister loci segregate independently of their timing of replication in an order roughly corresponding to their distance from parS sites [29], [41]. The results of this current study suggest that the segregation of chromosome I loci was no longer correlated to their genetic distance from parS1 sites when these sites had been displaced (Figures 3 and S44), but instead followed the replication program. The efficient segregation of V. cholerae chromosome I lacking parS1 sites suggests that parS1-polar mobilisation is not essential for the segregation process. The influence of the replication program on the organization and segregation of chromosome I was further assessed by inserting an ectopic origin of replication in the left replichore. The segregation and the organisation of the bulk of chromosome I was not altered if the partition machinery remained intact (Figure 5). However, when combined with the deletion of the parS1 sites, the addition of an ectopic origin of replication affected the positioning of loci proximal to the two functional origins (Figures 5 and 4B). Taken together, these results suggest that the longitudinal organization of chromosome I was preserved by the replication program when its partition system was disrupted (Figures 5 and 6A, ΔparS1).

In E. coli and B. subtilis, modification of the replication program by the addition or the displacement of the replication origin does not influence the organisation of the bacterial chromosome [40], [42]. In particular, replication initiation from an origin located in the middle of the right replichore did not disrupt the transversal organisation of the E. coli chromosome [40]. However, it increased the proportion of cells in which the origin sisters were displaced to the outer edges of the nucleoid, i.e. towards the cell poles, suggesting that replication program could contribute to polar mobilisation in E. coli. Moreover, the E. coli chromosome lost its transversal organisation and adopted a longitudinal organisation in mukB mutant cells [43], [44]. Preliminary studies suggest that deletion of the mukBEF genes in V. cholerae, which does not confer any loss of cell viability or any aberrant cell morphology (data not shown), does not alter the positioning and segregation of ori_I and L3_I (Figure S24). Nevertheless, future work is needed to investigate the contribution of the MukBEF system to the organization and segregation of the two V. cholerae chromosomes.

In conclusion, this study demonstrates that the partition machinery and the replication program contribute to the polar mobilization of the origin regions in V. cholerae. We hypothesise that it is the genomic proximity of parS sites to the oriC site, which is a conserved feature of most bacterial chromosomes that serves to ensure the convergence of their polar positioning activities.

Materials and Methods

Plasmids and strains

Bacterial strains and plasmids used in this study are listed in Tables S1 and S2 respectively. All V. cholerae mutants were constructed by integration-excision or natural transformation (Protocols and details on the construction of each strain in Text S1). To this end, a derivative of the El Tor V. cholerae N16961 was rendered competent by the insertion of hapR by specific transposition [45]. Engineered strains were confirmed by PCR.

Fluorescence microscopy

Cells were observed in Minimal Media to have only a single copy of each chromosome after division. Protocols for Microscopy are detailed in Text S1. The snapshot images were analysed using the Matlab-based software MicrobeTracker [45], [46], see details for the analysis in Supplementary Text S1.

Supporting Information

Figure S1

Timelapses of ADV21 and ADV23. Acquisition was every 5 min and only timepoints of interest are shown. Green foci correspond to yGFP-ParB_pMT1 and red foci to LacI-mCherry.

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Figure S2

Cell size distribution of different strains studied. They are plotted against ADV24 (ori_I and L3_I) as reference.

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Figure S3

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV20 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S4

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV21 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S5

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV22 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S6

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV23 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S7

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV24 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S8

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV25 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S9

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV26 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S10

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV27 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S11

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV30 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S12

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV33 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S13

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV39 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S14

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV42 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S15

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV50 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S16

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV51 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S17

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV78 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S18

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV114 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S19

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV115 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S20

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV123 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S21

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV128 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S22

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV130 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S23

Duplication frequency and reconstituted segregation choreography as a function of cell length in ADV131 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S24

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP454 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S25

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP560 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S26

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP568 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S27

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP582 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S28

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP591 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S29

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP599 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S30

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP604 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S31

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP605 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S32

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP626 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S33

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP633 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S34

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP634 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S35

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP655 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S36

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP656 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S37

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP659 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S38

Duplication frequency and reconstituted segregation choreography as a function of cell length in CP708 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S39

Duplication frequency and reconstituted segregation choreography as a function of cell length in EPV213 strain (not realigned). Top panel: numbers of cells in each cell size interval (0.2 µm); Middle panel: proportion of cells with duplicated foci according to cell size for each tagged locus; Bottom panel: relative median, 1 quartile and 3 quartiles positions of each locus according to cell size elongation. The principle of cell orientation is indicated.

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Figure S40

Localisation of Ypet-ParB1 in WT, ΔparS1, parS1_{300 kb} , parS1_{650 kb}. Overlay of Phase contrast (red) and GFP images (green) showing representative cells of each strains grown in M9 Fructose Thiamine medium.

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Figure S41

Chromosome II positioning is not dramatically modified in ΔparS1 cells. (A) Circular map indicating the position of the different loci analysed in the different genetic backgrounds: WT (left) and Δ parS1 (right). (B) Reconstitution of the segregation choreographies, as in Figure 1C. For the WT choreography, strain cell sizes were realigned as in Figure 2. For the Δ parS1 choreography, the cell sizes of ADV128 were aligned to the cell size of ADV27 using the first cell size interval where ≥50% of cells contained 2 foci of ori_II and R1_II, respectively. (C) Relative distance between R1_II and ter_II loci measured in the cells containing only one focus of each, as in Figure 1D. The positions of these 4 loci follow the order of the genetic map, from midcell (oriII) to the new pole (terII). Note, that the variability of the terII locus positioning was increased in ΔparS1 cells.

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Figure S42

Functionality of the ectopic oriC1_{651 kb} origin of replication. (A) Marker Frequency Analysis of chromosome I in exponentially growing WT (ADV24) and oriC1_{651 kb} (CP626) cells. The presence of sharp peaks of equivalent heights at the two origins of strain CP626 indicates that they simultaneously fired at each round of replication. (B) Proportion of the different cell types in WT (ADV24) and in oriC1_{651 kb} (CP626).

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Figure S43

Presence of oriC1 site is essential within the origin region. After introducing an ectopic oriC1 using natural transformation with pPOS228, we attempted to delete the original Origin of replication of the chromosome I. Two strategies were performed: a direct deletion by replacing the oriC1 with a Rif resistance gene (pAD43), or an indirect deletion, by first replacing oriC1 by an oriC1 flanked by FRT sites (pAD44) and secondly deleting it by inducing a Flipase protein (pFLP2). When the second oriC1 was introduced in the middle of the replichore (near L3_I locus), we were unable to delete the original oriC1, even thought it was possible to replace it by a FRT-oriC1-FRT. It was however possible to delete this Origin when the second oriC1 was at only 50 kb from it.

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Figure S44

Proportion of the different cell types in WT (ADV24) and in parS1_{490 kb} (CP634).

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Table S1

Strains list.

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Table S2

Plasmids list.

(DOCX)

Text S1

Supplementary Material and Methods.

(DOCX)

Funding Statement

This work was supported by Agence Nationale pour la Recherche [ANR-09-BLAN-0258] - European Research Council under the European Community's Seventh Framework Programme [FP7/2007–2013 Grant Agreement no. 281590]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.