doi: 10.1126/sciadv.1501914. eCollection 2016 Apr.
A checkpoint control orchestrates the replication of the two chromosomes of Vibrio cholerae
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- PMID:27152358
- PMCID: PMC4846446
- DOI: 10.1126/sciadv.1501914
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A checkpoint control orchestrates the replication of the two chromosomes of Vibrio cholerae
Marie-Eve Val et al. Sci Adv..
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Abstract
Bacteria with multiple chromosomes represent up to 10% of all bacterial species. Unlike eukaryotes, these bacteria use chromosome-specific initiators for their replication. In all cases investigated, the machineries for secondary chromosome replication initiation are of plasmid origin. One of the important differences between plasmids and chromosomes is that the latter replicate during a defined period of the cell cycle, ensuring a single round of replication per cell. Vibrio cholerae carries two circular chromosomes, Chr1 and Chr2, which are replicated in a well-orchestrated manner with the cell cycle and coordinated in such a way that replication termination occurs at the same time. However, the mechanism coordinating this synchrony remains speculative. We investigated this mechanism and revealed that initiation of Chr2 replication is triggered by the replication of a 150-bp locus positioned on Chr1, called crtS. This crtS replication-mediated Chr2 replication initiation mechanism explains how the two chromosomes communicate to coordinate their replication. Our study reveals a new checkpoint control mechanism in bacteria, and highlights possible functional interactions mediated by contacts between two chromosomes, an unprecedented observation in bacteria.
Keywords: Vibrio; cell cycle; cholera; chromosome; multipartite genome; pathogens; plasmids; replication; replication initiation; secondary chromosome.
Figures
(A) Top: Genome structure of wild-type (WT)V. cholerae. Ovals indicate the origins of replication (ori1 andori2) and triangles showdif sites (dif1 anddif2) on Chr1 (green) and Chr2 (red). Bottom: MFA of exponentially growing WT cultures using a corrected reference sequence of Chr1 (fig. S1). Log2 of number of reads starting at each base (normalized against reads from a stationary phase WT control) is plotted against their relative position on Chr1 and Chr2. Positions ofori1 andori2 are set to 0 for a better visualization of the bidirectional replication. Any window containing repeated sequences is omitted; thus, the large gap observed in the right arm of Chr2 consists of filtered repeated sequences within the superintegron (28). Green (Chr1) and red (Chr2) dots indicate the average of 1000-bp windows; black dots indicate the average of 10,000-bp windows. Dark green, light green, red, and orange lines indicateori1,ter1,ori2, andter2 number of reads, respectively; dashed blue lines indicate the Chr1 RctB binding locus (12). The same color code is used for all MFA figures. (B andC) Genomic variants CSV2 (Chr1 = 2.5 Mbp and Chr2 = 1.5 Mbp) (B) and ESC2 (Chr1 = 2 Mbp, Chr2 = 2 Mbp) (C) are the same as in (A). The genetic exchanges made between Chr1 and Chr2 are shown in green and red. (D) Chr1 map of WT and genomic variants (JB392, JB590, JB659, JB771, and JB963) with large chromosomal inversions around a fixed locus (VC018) and other loci located at increasing distances fromori1 (VC392, VC590, VC659, VC771, and VC963), respectively. For each genomic mutant, the loci flanking the DNA inversion are shown in red. The left (dark green) and right (light green) replichores are separated byori1 (oval) anddif1 (triangle). The position of the Chr1 RctB binding locus is indicated by a blue star. (E) Histogram representing quantitative PCR–measuredori1/ori2 ratios from relative gDNA quantification of exponentially fast-growing strains (gDNA from WT stationary culture was used for normalization). Bars display means (±SD) of at least three experiments. (F) Log2(ori1/ori2) plotted as a function of the distance betweenori1 and the RctB binding locus displays a linear relationship (R2 = 0.92). Dots show means (±SD) of three experiments.
(A) Circular map of WT Chr1 showing the native location ofcrtS (blue bar) and the various loci wherecrtS was relocated (gray bars) with respect toori1 (oval) anddif1 (triangle). The inside scale designates DNA size in kilobase pair. (B) MFA of relocatedcrtS mutants (crtSVC23, crtSVC392, crtSVC963, and crtSVC2238). The dashed blue lines indicate the number of reads of the loci wherecrtS has been relocated. (C) Log2(ori1/ori2) and log2(ori2/crtS) plotted as a function of the distance betweencrtS andori1 in kilobase pair. The ratios were calculated as the ratios of the number of reads per base pair (from MFA) for each designated loci.
(A andB) Plot showing the position ofori1 (left panel) andori2 (right panel) foci inside WT (A) and mutant crtSVC23 (B) cells. Foci are oriented longitudinally relative to the old pole of the cell as a function of cell length. The old pole of the cells was defined as the closest pole to anori1 focus. Thex axis represents the cell length (in micrometers). They axis represents the relative position of the focus in bacterial cells, 0 being the old pole and 1 the new pole. Snapshot images of 585 WT and 2072 crtSVC23 mutant cells were analyzed. (C andD) Histograms displaying the amount of cells that exhibit zero, one, two, three, four, or five and six fluorescent foci according to cell size (in micrometers) in WT (C) and mutant crtSVC23 (D) cells. (E andF) By correlating the longitudinal position ofori1 andori2 foci as a function of cell length, the segregation choreographies of theori1 andori2 were reconstituted throughout the cell cycle of WT (E) and mutant crtSVC23 (F) bacterial cells. Cells were classified according to their size and grouped by 30 to define each size interval. For most loci and cell length intervals, there were cells with either a single focus or two separated foci, the relative proportions of each type varying as a function of cell length. Only the position of the foci corresponding to the dominant cell type in each cell length interval was plotted. The median positions of the observed foci (filled circles), along with the 25th to 75th percentiles (error bars), were plotted for each cell size bin. Thex axis represents the cell length (in micrometers). They axis represents the relative position of the focus in bacterial cells (0, new pole and 1, old pole).
(A andB) Position ofori1 (A) andori2 (B) foci inside crtSWT/VC23 cells. Foci are oriented longitudinally relative to the old pole of the cell as a function of cell length. The old pole of the cell was defined as the closest pole to anori1 focus. On the left panel, thex axis represents the cell length (in micrometers). On the right panel, thex axis indicates cell number. They axis represents the relative position of the focus in bacterial cells, 0 being the old pole and 1 the new pole. (C) Representative pictures of dividing cells (WT and crtSWT/VC23) observed by fluorescence microscopy. Cells were fluorescently labeled nearori1 andori2. Merged pictures ofori1 (green) andori2 (red) and phase-contrast (blue) micrographs show 2× more red spots than green spots in mutant crtSWT/VC23. (D andE) Amount of crtSWT/VC23 (D) and crtSWT/VC2238 (E) cells exhibiting zero, one, two, three, four, or five and sixori1 foci (left panel) andori2 foci (right panel) according to cell size (in micrometers).
(A) Normalized and filtrated genomic contact map obtained from an asynchronous population of WT cells growing exponentially in LB.x andy axes represent genomic coordinates of each chromosome centered ondif sites (light green bar,dif1; orange bar,dif2). Origins of replication are shown as a dark green bar (ori1) and a dark red bar (ori2). Chr1 and Chr2 are represented by dark green (right arm) or light green (left arm) and dark red (right arm) or light red (left arm). The color scale reflects the frequency of contacts between two regions of the genome (arbitrary units), from white (rare contacts) to dark red (frequent contacts), and is conserved across all panels of all figures. (B) Interchromosomal contact map centered ondif (left panel) orori sites (right panel). ThecrtS site is indicated as a blue bar. (C andD) Circos representation of interactions of 100 kbp (20 bin) arounddif2 with Chr1 (C) and circos representation of interactions of 50 kbp (10 bin) aroundori2 with Chr1 (D).
(A) Phenotype ofcrtS-deleted mutants in WT and ICO1. Representative pictures of phase-contrast microscopy of live cells growing on LB agar pads: WT, ΔcrtS #7 (with two separate chromosomes), ICO1, and ICO1ΔcrtS. (B) MFA of WTΔcrtS #7. (C) Representative picture of a filamentous ΔcrtS cell observed with fluorescence microscopy. Loci nearcrtS (VC783) andori2 were fluorescently labeled. Merged pictures of VC783 (green) andori2 (red) and phase-contrast (blue) micrographs show a higher number of green spots than red spots. (D) Ethidium bromide–stained pulsed-field gel electrophoresis (PFGE) of native gDNA. From left to right: Independent clones ofcrtS-deleted mutants (WTΔcrtS #1 to #7), mutants with relocatedcrtS (crtSVC23, crtSVC392, crtSVC963, and crtSVC2238), and WT (two chromosomes) and MCH1 (one synthetic fused chromosome), which are used as size reference (18). (E) Same as (D). From left to right: WT-EV (control), MCH1 (one synthetic fused chromosome), and independent clones of ΔcrtS mutants before (D185, D247, C667, and C926) and after a 200-generation evolution (-EV). For C926, samples were harvested after 100 (-EV1) and 200 generations (-EV2) to track the tendency of the fused chromosome to revert to two separate chromosomes.
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