A Genetic System to Change Chromosome Configuration

By using the site-specific recombination system of bacteriophage λ, we previously developed a genetic system that allows the construction and detection of genetic inversions in the E. coli chromosome [25]. We have constructed several series of strains containing one defined att site at a fixed position and its att partner site inserted at random locations; strains carrying combinations of partner att sites that could give rise to viable recombinants have been selected. Cassettes were designed to detect inversion between att sites: recombination between attL and attR restores lacZ integrity (Figure 1B). By providing a limiting amount of recombinase, we were able to reveal the existence of MDs that correspond to large regions that are insulated from each other in the cell (Figure 1A). By providing a high amount of recombinase, recombination between most of the combinations of att sites can be detected, and there is a good correlation between the frequency of collisions and the frequency of recombinants [25]. There was no correlation between the frequency of inversion and the physiological properties of cells with inverted configuration; inversions occurring at high frequency can be detrimental, whereas those occurring at low frequency can be neutral (see below). We now analyze in detail the properties of strains carrying chromosomes with the different inverted configurations.

To unravel the consequences of inverting a chromosomal segment on cell physiology, we performed a number of analyses aimed at detecting defects visible at the colony or cell level. The size of colonies from strains with the inverted chromosome (lacZ reconstituted, blue colonies) was compared to that of strains with the wt configuration (white colonies) in rich medium. The effect of these inversions on growth was also measured using a coculture assay in which strains with chromosomes in wt and inverted configurations were compared (Materials and Methods). To analyze the consequences of the inversion on the nucleoid morphology, cells grown in exponential phase were stained with DAPI, and nucleoids were observed by fluorescence microscopy (see Materials and Methods). The percentages of cells with different types of nucleoids were numbered according to the cell size. The number of chromosome origins was estimated by fluorescence-activated cell sorting (FACS) analysis. Viability of strains was tested in different genetic backgrounds affected in pathways related to DNA metabolism. We used recA mutants and since RecA is required for both homologous recombination and SOS induction, the requirement of SOS induction for viability was tested in lexA ind⁻ (SOS⁻ Rec⁺) and recA lexA^def (SOS⁺ Rec⁻) mutants. When RecA was required, we used mutants affected in the two RecA-dependent recombination pathways, i.e., RecBC and RecFOR, to identify the pathway involved. Measurements of SOS induction were performed in a sfiA background to avoid SfiA-dependent filamentation and inhibition of cell division [39]. SOS induction was quantified in culture by using a plasmid carrying the uidA gene encoding β-glucoronidase under the control of the P_sfiA promoter (see Materials and Methods). In addition, the presence of a plasmid carrying a gfp gene under the control of the P_SfiA promoter allowed the direct visualization of the induction of the SOS response at the cellular level. tus mutants were used to estimate the defects provoked by inverted Ter sites in various configurations. When recombinant colonies could not be obtained, PCR reactions probing the presence of recombination at the DNA level were used to check for the occurrence of attL–attR inversion and for the presumed lethality conferred by the inversion.

Ter Sites Impede Replication Forks with Various Efficiencies

Ten Ter sites are found in the E. coli chromosome, which are bound in vitro by Tus with varying efficiencies [14]. The K_obs for Tus binding to the very strong TerB site is about 5 × 10⁻¹³ M, and the relative arrest activity was estimated to be around 95%. Although studies were not performed with other Ter sites, the effect of mutations in TerB mimicking the sequence of other Ter sites allows estimation of their respective strength as deduced from Tus–Ter binding affinity measurements and from measure of the replication arrest activity [14]. TerA–E and TerG are predicted to be very strong sites (arrest activity greater than 50%), TerH moderately strong (arrest activity around 33%), and TerF and TerI–J weak sites (arrest activity less than 20%). To estimate the respective strength of Ter sites in vivo in a strain producing wt levels of Tus, we have generated strains in which different Ter sites are inverted, and their properties have been analyzed (Figures S1 and S2, and Text S1). Altogether, the results indicate that efficiency of replication arrest at different chromosomal Ter sites correlates with the predictions based on in vitro affinities and on replication arrest activity of TerB mutant sites. They show that in conditions of wt level of Tus protein, blocking the two forks by the strong TerE and TerA sites renders RecBCD-dependent recombination essential for viability, as previously observed with TerA [15]. SOS induction is also essential in these conditions. The effect of the moderate site TerH in the inverted orientation was less severe, but still significant. Inverted weak TerI and TerJ sites do not appear to affect growth, suggesting they do not significantly impede replication (Figure S1 and Text S1).

Imbalance of Replication Arms Renders RecBC Essential for Growth

On the E. coli chromosome, the two replichores are of similar size, suggesting that most replication forks meet within the replication fork trap diametrically opposite to the origin. To evaluate the requirements for the balance of replication arms, we analyzed strains in which inversion endpoints are in each replication arm, asymmetrically relative to oriC (interreplichore inversion [Inter], Figure 2A, Table 1). As the inverted region contains oriC, these inversions do not change the orientation of sequences or genes relative to replication. However, because the two endpoints are at different distances from oriC, the size of the replication arms are modified, one becoming greater and one smaller than 50% of the chromosome. Imbalance of 5%–10% for replication arms has no effect on colony morphology: the colonies with inverted configurations are similar to those with wt configuration (Figure 2B, 47% for the short arm and 53% for the long arm (47–53) in strain Inter R-L3 (Table 1), and 42–58 in strain Inter R-L5 (Table 1)). The effect of these inversions on growth was also measured using the coculture assay containing strains with either a chromosome in wt or inverted configuration: no defect was associated to this genetic rearrangement as the ratio of inverted to wt cells was close to one after 60 generations (Figure 2C and unpublished data). The cells and nucleoids of strains with either configuration were not distinguishable (Figure S3). When the imbalance reached 15% (36–64 in Figure 2B, strain Inter R-NS^left1 in Table 1), some defects became apparent. The recombinant colonies were smaller than noninverted colonies, and the ratio of inverted to wt cells after 60 generations was affected (0.13 ± 0.02), but the cells and nucleoids of the inverted configuration were similar to those of the wt configuration: only 2% of the cells appeared abnormal (Figure 2D). Around 20% of imbalance (30–70 in Figure 2B, strain Inter R-NS^left2), the size of colonies carrying the inversion was affected; in coculture assays, the ratio of cells with inversion to wt configuration was less than 0.01 (Figure 2C) and microscopic observation showed longer cells with abnormal nucleoids (14% of abnormal cells in Inter R-NS^left2, Figure S3). Above 20% of imbalance (23–77 and 18–82 in Figure 2B, strains Inter R-NS^left4 and Inter R-O1, respectively, in Table 1), colonies were barely visible, and more than 20% of cells displayed condensed nucleoids, i.e., a par phenotype, or grew as cells with unsegregated nucleoids (Figure 2D and Figure S3, respectively).

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Figure 2. Imbalance of Replication Arms

(A) Schematic representation of the E. coli chromosome after interreplichore inversion leading to a 20% imbalance (replication arms are 30% and 70% of the total length of the chromosome). The two replication arms are indicated by red arrows, and the replication fork trap is indicated in yellow. The ten Ter sites (from A to J), replication origin oriC, and dif site are indicated.

(B) Colonies of strains carrying chromosomes with various levels of imbalance as indicated (47–53: strain Inter R-L3; 42–58: strain Inter R-L5; 36–64: strain Inter R-NS^left1; 30–70: strain Inter R-NS^left2; 23–77: strain Inter R-NS^left4; and 18–82: strain Inter R-O1). Colonies were obtained in a wt, recA, or recA tus genetic background.

(C) Quantification of the growth defects caused by replication arms imbalance. Strains carrying a chromosome with 3% (47–53), 14% (36–64), or 20% (30–70) of replication arms imbalance described in (B) were grown in serial cocultures with the strain carrying the wt configuration. The ratio of inverted to wt configurations is plotted as a function of the number of generations.

(D) Comparison of microscopic analyses of strains carrying a chromosome with 14% (36–64) or 32% (18–82) of replication arms imbalance described in (B). Colored horizontal bars indicate the percentage of the different types of cells and nucleoids in the wt (noninverted; left) and inverted (right) configurations. Green indicates cells containing one and two nucleoids (1+2 nuc); yellow: cells containing four nucleoids (4 nuc); cyan: cells containing par-like nucleoids (par); red: cells with unsegregated nucleoid (CUN); and pink: anucleate (anuc).

https://doi.org/10.1371/journal.pgen.0030226.g002

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Table 1.

Strains with Rearrangements Generating Inverted Ter Sites, Imposing Imbalance of Replication Arms, and Affecting Gene Orientation

https://doi.org/10.1371/journal.pgen.0030226.t001

Interestingly, we noticed that all strains with an imbalance greater than 20%, i.e., with a replication arm smaller than 30% and the other larger than 70%, were dependent on RecA for viability (Figure 2B and Table 1). Recombinant colonies could be obtained in a recFOR background, but not in conditions inhibiting either RecBC DNA recombination or SOS induction, indicating that the RecBC homologous recombination pathway is required for viability in the presence of an imbalance of replication arms greater than 20% (Table 1). The dependence on RecA for viability was suppressed by a tus deletion, indicating that the impediment of replication forks by Tus at Ter sites is responsible for lethality in a recA background (Figure 2B). Finally, a 2- to 4-fold SOS induction was apparent in strains that required recA for viability (Table 1). The analysis performed with inverted Ter sites indicated that in cells expressing wt levels of Tus, the replication forks are stopped at the first strong Ter site in the nonpermissive orientation ([15] and Text S1). It implies that, when the imbalance is smaller than 20%, the two forks of a same replication round can progress to the replication fork trap. In contrast, RecBC-dependent recombination is solicited to restart the first fork that reaches a Ter site before the other fork can reach it when the imbalance of replication arms is larger than 20%. We propose that, in the conditions used, when the shorter replication arm is less than half the longer one, it is fully replicated twice before completion of replication of the longer arm, leading to the formation of DNA double-stranded ends. These double-stranded ends induce the SOS response and are lethal in the absence of RecARecBC-dependent homologous recombination. Many natural inversions in bacterial genomes are symmetrical with respect to replication origins and termini. Scatter plots of the conserved sequences between related species produce an X-shaped pattern, called X-alignment [2]. These rearrangements reveal that selection operates to maintain replichores of similar lengths; in most genomes, the size of the longest predicted replication arm does not exceed 60% of the chromosome [13]. By moving the position of the replication fork trap on the genetic map, we have been able to analyze the effect of varying the imbalance of replication arms. Remarkably, we did not observe negative effects when the imbalance was around 10%, in total agreement with the observed size distribution of replichores in different species. Some defects appeared when the imbalance reached 15%, and recombinational rescue of replication forks was required above 20%.

The analysis of interreplichore inversions affecting at the same time two MDs revealed that making hybrid MDs while keeping the wt replichore junction unaffected was well tolerated (Figure S3). We noticed that for similar levels of imbalance less than 20%, inversions involving endpoints located either in the NS regions or in the Left, Right, and Ter MDs (Figure S3 and Table 1) behave similarly: the growth of recombinant colonies was slightly affected, and recombinants were viable in a recA background (Table 1). It is only when the imbalance exceeded 20% that recombinant colonies were affected and their formation recA-dependent (Table 1). Altogether, these results suggest that in the context of interreplichore inversions, the effect of MD disorganization for the Left, Right, and Ter MDs can be well tolerated by the cell.

Large Intrareplichore Inversions between NS Regions and MD Are Not Detrimental for Growth

We noticed that large inversions inside a replichore (intrareplichore) with one endpoint in the Ori MD and the other in the NS^right region gave rise to recombinants with no strong defects. Three examples of strains with such rearrangements are shown in Figure 3. These inversions encompass 916, 927, and 668 kb corresponding to 826, 828, and 607 genes, including four, three, and one rDNA operons, respectively (Figure 3A, strains Intra O-NS^right1 to −3 in Table 1). Similar outcomes were obtained in the left replichore. For example, the inversion of a 982-kb–long segment that changes the orientation of 942 genes, including two rDNA operons and 34 ribosomal protein genes (strain Intra L-NS^left1 in Table 1) had no detectable detrimental effects (Figure 3B and unpublished data). The colonies of strains with the rearranged chromosome had the same size as those with the wt configuration (Figure 3B). The diagram shown in Figure 3C indicates that even the largest inversion has no detectable effect on nucleoid morphology. No strong defect was associated with these genetic rearrangements, because the ratio of inverted to wt configuration was above 0.75 after 60 generations in coculture assays (Figure 3D). Finally, colonies carrying these inverted configurations were viable in a recA background, indicating the absence of important DNA damage (Figure 3B). Altogether, these results indicate that the direction of replication can be inverted through hundred of genes, including rDNA genes, without important consequences for growth. Furthermore, the results show that inversions between Ori MD and the NS regions are well tolerated. Similar conclusions were obtained from the analyses of intrareplichore inversions between NS regions and the flanking Right or Left MD in the absence of active Ter sites (Figure S4 and Table 1). Therefore, gene orientation, gene dosage, and sequence skews appear to operate only as long-term positive selection determinants. Our results are in agreement with the evidence [11,40] that weakens the proposed influence of replication on gene orientation [41,42]. However, given the large size of bacterial populations, slightly deleterious effects that can be accredited to positioning rDNA and ribosomal protein genes on the lagging strand are most likely sufficient to eliminate such configurations from the population in long-term evolution.

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Figure 3. Tolerance to Large Inversions within a Replichore

(A) Schematic representation of the E. coli chromosome showing the MDs (Ori in green, Right in red, Left in blue, and Ter in cyan), the seven rrn operons (rrsA to rrsH), replication origin oriC, migS, and dif. The numbered black arcs indicate the position and extent of fragments inverted in the strains 1 to 4 (1: intra O-NS^right1; 2: intra O-NS^right2; 3: intra O-NS^right3; and 4: intra O-NS^left1).

(B) Colonies of strains 1 to 4 carrying normal and inverted configurations. Colonies were obtained in a wt or recA genetic background.

(C) Comparison of microscopic analyses of cells from strain 1 in wt (noninverted) and inverted configurations as described in Figure 2.

(D) Quantification of the growth defects caused by intrareplichore NS^right–Ori inversions. Strains 1 to 3 carrying the inverted configuration were grown in serial cocultures with the strain carrying the wt configuration. The ratio of inverted to wt configurations is plotted as a function of the number of generations.

https://doi.org/10.1371/journal.pgen.0030226.g003

In contrast to well-tolerated inversions described above, two types of intrareplichore inversions were highly detrimental for the cell: the first type involved endpoints located in the Ter and the Right MDs, and provokes the separation of the replication fork trap from the wt replichore junction. The second type involved inversion between endpoints located in the Ori MD and in the Right MD. Features of these two detrimental configurations are described in detail below.

Intrareplichore Inversions That Disrupt the Ter Macrodomain Are Deleterious

Intrareplichore inversions with endpoints in the Right and Ter MDs (Figure 4A, strains Intra R-T1 to −3 in Table 2) generate a hybrid Right-Ter MD in which the orientation of TerA, TerD, and TerE is modified, creating a replication arms imbalance close to 35%–65% (see intra R-T1 in Figure 4B). These strains carry two zones of converging KOPS (Figure 4C): the normal one corresponding to the wt replichore junction, and a new one associated with the replication fork trap in the hybrid Right-Ter MD. Inversion severely affected the growth of colonies (Figure 4D). The observation of cells with the inverted configuration revealed the occurrence of a high proportion of abnormal cells: 27% of cells showed a par phenotype, 15% formed cells with unsegregated DNA, and 1% of cells were anucleate (strain Intra R-T1 in Figures 4E, 4F, and S5). Cells larger than 10 μm with a high amount of nonsegregated nucleoids were observed. FACS analyses indicated that the number of chromosomes in the large cells ranged from 16 to 32 (unpublished data). Other strains with intrareplichore inversions between Right and Ter MDs (Intra R-T2 and −3 in Figure 4 and Table 2) showed the same features (unpublished data).

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Figure 4. Intrareplichore Inversions between the Right and Ter MDs

(A) Schematic representation of the E. coli chromosome showing the MDs (Ori in green, Right in red, Left in blue, and Ter in cyan), the ten Ter sites (from A to J), oriC, and dif. The numbered red arcs indicate the position and extent of fragments inverted in the strains Intra R-T1 to −3 (1 to 3, respectively), and the pink arc (number 4) shows the fragment inverted in the Inter R-T2 strain.

(B) Schematic representation of the E. coli chromosome upon an intrareplichore inversion that causes the intermingling of Right and Ter MDs (strain Intra R-T1). The MDs are colored as in Figure 1, and the replication fork trap is indicated in yellow. The different Ter sites, oriC, and dif are indicated.

(C) Linear genetic maps of the Right and Ter MDs upon various intrareplichore inversions. The inverted Ter site defining the displaced replication fork trap is shown, and the replication fork trap is indicated in yellow. The dif site is indicated by a small stick, and the wt replichore junction by a grey box. Inverted KOPS are indicated by rafters (1 for 100 kb). Viability in a recA background is indicated by a plus sign (+) beside the map.

(D) Colonies of strains carrying chromosomes with various intrareplichore inversions as indicated in (C). Colonies were obtained in a wt or recA genetic background.

(E) Microscopic analysis of strain Intra R-T1 as described in Figure 2.

(F) Cells from strain Intra R-T1 with an inverted configuration were transformed with a plasmid expressing gfp under the P_sfiA promoter. Nucleoids of cells expressing SOS appear as blue (see Figure S3 for unmerged pictures).

(G) Pathways required for viability of the inverted configuration. Genetic backgrounds in which inverted configuration did not give rise to viable colonies are indicated in red.

https://doi.org/10.1371/journal.pgen.0030226.g004

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Table 2.

Strains with Rearrangements Making Hybrid Right–Ter MDs and Ori–Right MDs

https://doi.org/10.1371/journal.pgen.0030226.t002

The origin of the detrimental phenotypes caused by this chromosomal configuration was analyzed by testing different genetic backgrounds (Figure 4G). It was not possible to obtain viable recombinants in a lexA ind⁻ background, i.e., in SOS-defective conditions. SOS induction was directly visualized by the use of a plasmid expressing gfp under the control of P_SfiA promoter (Figures 4F and S5). Homologous recombination was also required because recombinants with the inverted configuration could not be obtained in a recA, recBC, or in a recA lexA^def background (i.e., in conditions of constitutive SOS induction, but in the absence of RecA-dependent recombination).

The phenotype and RecA-independence of interreplichore Right-Ter inversions (Figure S3, strains Inter R-T1 to −4 in Table 1) suggests that intermingling Right and Ter MDs cannot by itself be responsible for the growth defects of strains Intra R-T1 to −3 in the inverted configuration. Growth defects and RecA dependence for viability were suppressed in a tus background, indicating that the position of the displaced replication fork trap is responsible for the growth defects (Figure S5E). The detrimental effects can not originate only from imbalance of replication arms because the imbalance of replication arms is close to 35–65, a level that does not render RecA essential for viability in interreplichore inversions (Figure 2 and Table 1). Three other hypotheses that might account for the growth defects were tested below: the positioning of dif outside of the replication fork trap, the presence of two zones of converging KOPS, and the merging of replication forks far away from the wt replichore junction region.

In these intrareplichore Right-Ter inversions, the replication fork trap is separated from dif. It was previously reported that the dif site does not need to be present in the replication fork trap to be fully active because the insertion of a ectopic TerA* site near TerA, moving the replication fork trap away from the dif region, did not affect dif activity [43]. dif is active in any new replichore junction formed after inversion [9,27]. After deletion of dif from its normal position, we reinserted a 28-bp fragment corresponding to dif in the new replication fork trap, in the region where KOPS converge, far away from the wt replichore junction (Figure 4C, strain Intra R-T2 Δdif dif^RFT in Table 2). Strains carrying this inverted configuration still showed strong detrimental defects and were not obtained in a recA background (Table 2) even though insertion of dif in the new replication fork trap improved nucleoid distribution in a way suggesting dif activity, i.e., by removing a 12%–15% fraction of filaments (47% of abnormal cells instead of 64% in the absence of dif, and 50% when dif is present at its normal location; unpublished data). These results indicate that the absence of dif from the new replication fork trap is not responsible for the observed defects. These results were corroborated by the viability of Inter Right-Ter inversions in a recA background when dif was deleted (strain Inter R-T2 Δdif in Table 2), confirming that the RecA dependence for viability of Intra Right-Ter inversions does not result from the lack of dif in the replication fork trap.

To analyze the defects provoked by forming two zones of converging KOPS and by positioning the replication fork trap far from the dif region but without Right and Ter MDs intermingling, we constructed strains with an inversion that positioned the replication fork trap at the limit between the Ter and the Right MDs (Intra T1 in Figure 4C, and strains Intra T1 and Intra T2 in Table 2). Colony formation was not affected; cells and nucleoids were similar to those of the wt configuration, and strains carrying inversions were viable in a recA background (Figure 4C and 4D). These results indicate that as long as sequences belonging to the Ter MD remain together, merging of replication forks far away from the wt replichore junction in the presence of two zones of converging KOPS does not provoke important growth defects.

To determine whether merging of replication forks outside the Ter MD may be responsible for the detrimental effects of intrareplichore Right-Ter inversions, we generated two different genetic inversions in the Right MD (strain Intra R3 in Figure 4C, and strains Intra R3 and Intra R4 in Table 2) that inverted TerE in a strain in which TerA and TerD are deleted; inversion of the TerE region provoked replication to end in the Right MD, and generated two zones of converging KOPS (Figure 4C) and an imbalance of replication arms close to 35–65 (Table 2). Recombinant colonies were slightly affected compared to those with a wt configuration (Figure 4D); cells and nucleoids from both configurations were similar (unpublished data), and recombinants were viable in a recA background (Figure 4D). These results indicate that replication forks can merge in the Right MD without affecting viability.

Because none of the simple modifications in the chromosome structure can, by itself, account for the growth defect of intrareplichore Right-Ter inversions, we tested whether the defect was dependent on the length of the Right MD that separates the replication fork trap from the wt replichore junction in the Intra R-T1 configuration. We constructed strain Intra R-T4 (Figure 4C and Table 2). In this strain, the chromosome configuration is similar to Intra R-T1, Intra R-T2 and Intra R-T3 configurations, but the extent of sequences belonging to the Right MD that are embedded in the Ter MD is reduced (170 kb compared to 420 kb). Recombinants showed fewer defects; only a fraction of cells (13%) showed a par phenotype, and less than 1% formed cells with unsegregated nucleoids (Figure S6). Importantly, strains in the inverted configuration were viable in a recA background (Figure 4D). These results are in agreement with the hypothesis that the extent of Right MD DNA that separates the Ter region where fork merge from the replichore junction region is responsible for the observed defects. The combination of the embedding of Ter sequences in the Right MD and finishing replication within these Ter sequences is responsible for the deleterious effect. The shortening of the region of Right MD that separate the replication fork trap from the wt replichore junction region suppresses the defects. Together with the observed viability of recA- interreplichore inversions involving the Right and the Ter MDs (strains Inter R-T1 and R-T2 in Figure S3 and Table 1), these observations support the hypothesis that the requirement of RecA for the viability of intrareplichore Right-Ter inversions results from the separation of the replichore junction region from the region in the Ter MD where replication ends. It is therefore tempting to speculate that in deleterious configurations resulting from intrareplichore inversion, replication ends in the displaced part of the Ter MD, activities normally associated to the wt replichore junction region cannot be performed, and the cell cycle is affected. Altogether, these results suggest the existence of a tight temporal and/or spatial coupling between the end of DNA replication in the Ter MD and an unknown activity near the replichore junction region required to progress in the cell cycle. Further work will be required to determine whether proteins known to function near the terminal replichore junction, FtsK [44] and TopoIV [45], are involved in this coupling.

Intrareplichore Inversion That Intermingle Ori and Right MDs Are Detrimental

The second class of detrimental inversions corresponds to intrareplichore inversions that combine Ori MD with the Left or Right MD. Because most of the inversions between the Left and Ori MDs also induce a high imbalance of replication arms, we focused our study on the inversion between Ori and Right MDs. The detrimental effects of intermingling Ori and Right MDs were revealed by combining attL and attR sites inserted at various positions in the Right (14′, 17′, 19′, and 22′) and Ori (0.7′, 97′, 95′, 94′, 92′, 88′, 87′, and 86′) MDs (Figure 5A, Table 2, and unpublished data). Viable recombinants could be obtained only when the inversion involved sites located between 92′ and 0.7′ in the Ori MD, and they all showed strong growth defects (proximal Ori–Right combinations, strains Intra O-R1 to −5 in Figure 5A [indicated in grey] and 5B). In contrast, viable recombinants could not be obtained when the inverted fragment extended from the Right MD to 88′, 87′, or 86′ (distal Ori-Right combinations, strains Intra O-R6 to −8 indicated in black in Figure 5A). Proximal inversions invert the TerHI sites as inversions between Right MD and NS^right region (control c in Figure 5A, Intra R-NS^right3 in Table 1), whereas distal inversions invert both TerHI sites and the centromere-like sequence migS found at 89′. migS did not seem to be responsible for the difference observed between the two types of combinations since distal combinations did not give rise to viable recombinants in the absence of migS (unpublished data). In the absence of both TerH and TerI (ΔTerHI in Figure 5C), viable recombinant colonies with no strong growth defects could be obtained for proximal inversions, and they are viable in a recA background (strains Intra O-R1 to −3 in Figure 5C and Text S1). Distal inversions remained lethal on rich medium when both TerH and TerI were deleted (Figure 5D and unpublished data, and strains Intra O-R6 to −8 in Table 2), but viable colonies could be obtained on minimal growth medium (Figure 5D). These recombinants showed growth defects in minimal medium supplemented with casamino-acids (Figure 5D) and could not be propagated in rich medium (unpublished data).

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Figure 5. Intrareplichore Inversions between the Right and Ori MDs

(A) Schematic representation of the E. coli chromosome showing MDs, Ter sites, oriC, migS, and dif. The numbered black arcs indicate the position and extent of fragments inverted in strains 1 to 8 and (1 to 8: intra O-R1 to −8 in Table 2), and the control strain c is indicated in pink (control Intra R-NS^right3 in Table 1).

(B) Colonies of strains carrying chromosomes with various intrareplichore inversions as indicated in (B). Inversion tests for strains 1 to 8 were performed in rich medium. Colonies carrying the inverted configuration of strains 1 to 5 can be obtained in rich medium (indicated by a grey arc), whereas colonies carrying the inverted configuration of strains 6 to 8 cannot be obtained (indicated by a black arc).

(C) Colonies of strains 1 to 3, in the absence of TerH or TerH and TerI, carrying the chromosome in the wt or inverted configurations. Colonies were obtained in a wt or in a recA background. Also shown are the results obtained after deletion of the TerH site from strain c in a wt or recA genetic background.

(D) Colonies of strain 6 deleted for TerH and TerI, in the wt or inverted configuration, obtained in minimal medium (MM) or minimal medium supplemented with casamino-acids (MM casa).

(E) Microscopic analysis of strain 3 carrying a chromosome in the wt (noninverted) and inverted configurations as described in Figure 2.

(F) Nucleoid and cell analysis obtained from strain 3 deleted for TerH and TerI, carrying a chromosome in the wt or inverted configuration. The left and right colored horizontal bars indicate the percentage of the different types of cells and nucleoids in the wt and inverted configurations, respectively (green indicates cells containing one and two nucleoids; yellow: cells containing four nucleoids; cyan: cells containing par-like nucleoids; red: filamentous cells with apparently segregated nucleoids; and pink: anucleate cells).

(G) Images of combined DIC-DAPI and DIC-stained cells from strains 1 and 3 deleted for TerH and TerI, in the inverted configurations, showing the presence of filamentous cells with segregated nucleoids.

(H) Combined images of DIC and DAPI pictures of cells from strain 6 deleted for TerH and TerI, in the inverted (inv) or wt configuration after growth in rich medium for 0, 180, or 300 min.

(I) Nucleoid and cell analysis obtained from strain 3 deleted for TerH and TerI, carrying a chromosome in the wt or inverted configuration, after 300 min of growth in rich medium. The left and right colored horizontal bars indicate the percentage of the different types of cells and nucleoids in the wt and inverted configurations, respectively (green indicates cells containing one and two nucleoids; yellow: cells containing four nucleoids; and red: filamentous cells with un-segregated nucleoids).

https://doi.org/10.1371/journal.pgen.0030226.g005

The proximal Ori–Right inversions that gave rise to viable colonies were used for microscopy analysis (strains Intra O-R1 to −3 in Table 2). In the presence of TerH and TerI, we observed a predominant filamentation with DNA accumulating in nonsegregated nucleoids (e.g., 39% of filaments and 10% of par-like cells in the inverted configuration of Intra O-R3 in Figure 5E, and unpublished data). Analysis of the nucleoids of recombinant colonies deleted for TerH and TerI revealed a high percentage of normal cells. However, in the inverted configurations, a significant proportion of cells formed filaments (5%, 9%, and 22%, according to the strain, Figure 5F and 5G, and unpublished data). Remarkably, these filaments were different from those observed in all other rearrangements described in this study; they showed apparently segregated nucleoids with no division septa between DNA bodies (Figure 5G).

To visualize the defects responsible for the absence of viability in distal Ori–Right combinations in rich medium, cells obtained in minimal medium were grown in liquid rich medium and observed at different time points. After 180 min, cells with inverted configurations accumulated a fraction of abnormal cells (16% of filaments not observed in wt cells; Figure 5H and unpublished data), whereas after 300 min, most of the cells were elongated, with improperly compacted and segregated nucleoids (Figure 5H and 5I). Altogether, these results indicate that intermingling Right and Ori MDs interferes with nucleoid management and formation of the division septum. For both proximal and distal combinations involving Ori–Right MDs, the presence of longer cells with an increased number of segregated nucleoids indicates an inhibition in the formation of a septum of division. It is striking to note that inversions that move the Ori MD close to the Right MD (less than 50 kb from the Right MD; strain Intra O-NS^right4 in Table 1, Figure S7) slightly affect cell physiology. It is therefore likely that the origin of the defect results from an antagonism between Ori and Right MDs rather than from simply moving Ori sequences on the genetic map. The observation that distal inversions were more problematic than proximal ones suggests that the deleterious effects are proportional to the length of the MD. In B. subtilis, the chromosome partitioning and sporulation protein Spo0J binds eight parS sites scattered in the 800-kb region flanking oriC [46]. Our results suggest the presence of similar specific sequences in the Ori MD. Imbedding of such putative sequences in the Right MD could be the reason for growth defects. In this regard, it is interesting to note that the NS^right region separates Ori and Right MDs and could play a buffer role. We would like to speculate that mixing Ori and Right MD sequences would perturb proper segregation of Ori and Right MD, a step necessary to establish septum division. Further experiments will be necessary to determine whether the perturbation of the spatial control of cytokinesis affected by this type of inversion involves SfiA [39], MinCDE [47], SlmA [48], or other unidentified proteins.

Altogether, the results reported here give an important insight into the role of MDs in cell cycle control by chromosome configuration in E. coli (Figure 6). The Ter MD is involved in a process that spatially and/or temporally couples the end of replication in the Ter MD with a subsequent step near the replichore junction region. The antagonistic Ori and Right MDs are involved in a process coupling chromosome segregation and cytokinesis. Identification of determinants or factors that specify MDs should help us understand how MDs are involved in the control of these processes.

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Figure 6. Detrimental Inversions in E. coli

Schematic representation of the E. coli chromosome showing MDs, Ter sites, oriC, migS, and dif. The black arcs indicate the position and extent of fragments for which inversion is detrimental for growth. Grey arcs indicate inversions between Right and Ter or Right and Left MDs that are tolerated. Well-tolerated intrareplichore inversions between NS^right and adjacent MDs are not indicated. The analysis of detrimental and nondetrimental inversions supports a model in which the Ter MD is involved in a process that couples the end of replication in the Ter MD with a subsequent step. Inversion between Ori and Right MDs results in the perturbation of a process controlling chromosome segregation and cytokinesis.

https://doi.org/10.1371/journal.pgen.0030226.g006

Bacterial strains.

E. coli K12 strains are all derivatives of MG1655. Standard transformation and transduction procedures used were as described before [25]. Plasmids and strains with relevant genotypes are described in Table S1.

Induction.

Conditions for inversion formation were as described previously [25].

Detection and measurement of the SOS response.

SOS response was quantified by measuring the amount of β-glucoronidase [49] in sfiA cells transformed by a pBAD18-derived plasmid carrying the uidA gene fused to the sfiA promoter. Similar results were obtained in a sfiA⁺ background, but results were less variable in a sfiA background. SOS induction was visualized by using cells transformed by a P15A derivative carrying gfp under the control of the sfiA promoter (pZA-P_sfiA-gfp).

Microscopy and flow cytometry analyses.

The cultures were grown to optical density (OD) 0.2 at 30 °C and then processed for microscopy or flow cytometry. For the microscopy analysis, the cells were processed as described before [45]. For flow cytometry analysis, chromosome numbering was estimated by counting the number of replication origins using a rifampicin/cephalexin replication run-out [50]; aliquots were taken every 10 min over a period of 200 min. Cells were fixed in a 75% ethanol–PBS 1× solution, then washed in PBS 1×, treated with RNaseA, and the DNA was then stained with propidium iodide. The cells were analyzed on a Partec PASIII flow cytometer.

Coculture assay.

Strains carrying the inverted configurations were grown in coculture with the same strain carrying the wt configuration. A 1:1 mixture of the two strains was grown in serial cultures in LB medium at 30 °C for up to 70 generations. Every 10 generations, the relative numbers of both configurations were determined by plating. Experiments were performed in triplicate.