Research ArticleMICROBIOLOGY

A checkpoint control orchestrates the replication of the two chromosomes of Vibrio cholerae

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Science Advances  22 Apr 2016:
Vol. 2, no. 4, e1501914
DOI: 10.1126/sciadv.1501914
  • Fig. 1 Chr1 and Chr2 replication coordination is promoted by the presence of a timer on Chr1 and not by the requirement to terminate their replication synchronously.

    (A) Top: Genome structure of wild-type (WT) V. cholerae. Ovals indicate the origins of replication (ori1 and ori2) and triangles show dif sites (dif1 and dif2) 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 of ori1 and ori2 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 indicate ori1, ter1, ori2, and ter2 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 and C) 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 from ori1 (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 by ori1 (oval) and dif1 (triangle). The position of the Chr1 RctB binding locus is indicated by a blue star. (E) Histogram representing quantitative PCR–measured ori1/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 between ori1 and the RctB binding locus displays a linear relationship (R2 = 0.92). Dots show means (±SD) of three experiments.

  • Fig. 2 Timing of replication of the Chr1 RctB binding locus (crtS) controls the timing of initiation of Chr2.

    (A) Circular map of WT Chr1 showing the native location of crtS (blue bar) and the various loci where crtS was relocated (gray bars) with respect to ori1 (oval) and dif1 (triangle). The inside scale designates DNA size in kilobase pair. (B) MFA of relocated crtS mutants (crtSVC23, crtSVC392, crtSVC963, and crtSVC2238). The dashed blue lines indicate the number of reads of the loci where crtS has been relocated. (C) Log2(ori1/ori2) and log2(ori2/crtS) plotted as a function of the distance between crtS and ori1 in kilobase pair. The ratios were calculated as the ratios of the number of reads per base pair (from MFA) for each designated loci.

  • Fig. 3 Segregation of ori2 (but not ter2) occurs earlier when crtS is transposed near ori1.

    (A and B) Plot showing the position of ori1 (left panel) and ori2 (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 an ori1 focus. The x axis represents the cell length (in micrometers). The y 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 and D) 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 and F) By correlating the longitudinal position of ori1 and ori2 foci as a function of cell length, the segregation choreographies of the ori1 and ori2 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. The x axis represents the cell length (in micrometers). The y axis represents the relative position of the focus in bacterial cells (0, new pole and 1, old pole).

  • Fig. 4 Two chromosomal copies of crtS doubles Chr2 copy number.

    (A and B) Position of ori1 (A) and ori2 (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 an ori1 focus. On the left panel, the x axis represents the cell length (in micrometers). On the right panel, the x axis indicates cell number. The y 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 near ori1 and ori2. Merged pictures of ori1 (green) and ori2 (red) and phase-contrast (blue) micrographs show 2× more red spots than green spots in mutant crtSWT/VC23. (D and E) Amount of crtSWT/VC23 (D) and crtSWT/VC2238 (E) cells exhibiting zero, one, two, three, four, or five and six ori1 foci (left panel) and ori2 foci (right panel) according to cell size (in micrometers).

  • Fig. 5 Intra- and interchromosomal interactions in V. cholerae.

    (A) Normalized and filtrated genomic contact map obtained from an asynchronous population of WT cells growing exponentially in LB. x and y axes represent genomic coordinates of each chromosome centered on dif 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 on dif (left panel) or ori sites (right panel). The crtS site is indicated as a blue bar. (C and D) Circos representation of interactions of 100 kbp (20 bin) around dif2 with Chr1 (C) and circos representation of interactions of 50 kbp (10 bin) around ori2 with Chr1 (D).

  • Fig. 6 crtS is crucial for Chr2 replication initiation at ori2.

    (A) Phenotype of crtS-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 near crtS (VC783) and ori2 were fluorescently labeled. Merged pictures of VC783 (green) and ori2 (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 of crtS-deleted mutants (WTΔcrtS #1 to #7), mutants with relocated crtS (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.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/4/e1501914/DC1

    Materials and Methods

    fig. S1. Chromosomal inversion detected around ori1 when WT sequences are mapped against the National Center for Biotechnology Information (NCBI) reference genome AE003852.

    fig. S2. Large DNA inversions either caused no fitness cost (JB392) or were similarly affected (JB590, JB659, JB771, and JB963).

    fig. S3. Complementation of ΔcrtS filamentous phenotype by addition of an ectopic chromosomal copy of crtS.

    fig. S4. Duplication and segregation of VC783 and ori2 foci in WT and mutant crtSVC23 throughout the cell cycle.

    fig. S5. Duplication and segregation of ter1 and ter2 foci in WT and mutant crtSVC23 throughout the cell cycle.

    fig. S6. ori2 foci duplicate earlier when crtS is located near ori1.

    fig. S7. The addition of an extra copy of crtS affects the growth of V. cholerae.

    fig. S8. Doubling in ori2 copy number in mutant with two chromosomal copies of crtS.

    fig. S9. Comparison of global chromosome organization of V. cholerae in different growth conditions.

    fig. S10. Comparison of directional index analysis at a 100-kbp scale with transcription and GC content for fast-growing cells.

    fig. S11. ΔcrtS mutants have a fitness defect, whereas ICO1ΔcrtS shows no additional growth defect.

    fig. S12. Fitness improvement of ΔcrtS mutants by the acquisition of compensatory mutations.

    fig. S13. Loss of filamentation phenotype of ΔcrtS mutants by the acquisition of compensatory mutations.

    fig. S14. MFA of crtS mutants before and after acquisition of compensatory mutations.

    fig. S15. The effect of MFA normalizations.

    table S1. Compensatory mutations obtained after evolution of ΔcrtS mutants.

    table S2. List of plasmids and bacterial strains.

    table S3. Primers used in qPCR.

    movie S1. 3D representations of the contact map from fig. S9 (exponential growth of the WT in LB).

    movie S2. 3D representations of the contact map from fig. S9 (exponential growth of the WT in MM).

    movie S3. Time-lapse fluorescence microscopy of ΔcrtS filamentous cells, tagged at VC783 (Chr1) and near ori2 (Chr2), growing on an M9 MM agar pad supplemented with fructose and thiamine.

    movie S4. Time-lapse fluorescence microscopy of ΔcrtS filamentous cells, tagged at VC783 (Chr1) and near ori2 (Chr2), growing on an M9 MM agar pad supplemented with fructose and thiamine.

    References (5064)

  • Supplementary Materials

    This PDF file includes:

    • Materials and Methods
    • fig. S1. Chromosomal inversion detected around ori1 when WT sequences are mapped against the National Center for Biotechnology Information (NCBI) reference genome AE003852.
    • fig. S2. Large DNA inversions either caused no fitness cost (JB392) or were similarly affected (JB590, JB659, JB771, and JB963).
    • fig. S3. Complementation of ΔcrtS filamentous phenotype by addition of an ectopic chromosomal copy of crtS.
    • fig. S4. Duplication and segregation of VC783 and ori2 foci in WT and mutant crtSVC23 throughout the cell cycle.
    • fig. S5. Duplication and segregation of ter1 and ter2 foci in WT and mutant crtSVC23 throughout the cell cycle.
    • fig. S6. ori2 foci duplicate earlier when crtS is located near ori1.
    • fig. S7. The addition of an extra copy of crtS affects the growth of V. cholerae.
    • fig. S8. Doubling in ori2 copy number in mutant with two chromosomal copies of crtS.
    • fig. S9. Comparison of global chromosome organization of V. cholerae in different growth conditions.
    • fig. S10. Comparison of directional index analysis at a 100-kbp scale with transcription and GC content for fast-growing cells.
    • fig. S11. ΔcrtS mutants have a fitness defect, whereas ICO1ΔcrtS shows no additional growth defect.
    • fig. S12. Fitness improvement of ΔcrtS mutants by the acquisition of compensatory mutations.
    • fig. S13. Loss of filamentation phenotype of ΔcrtS mutants by the acquisition of compensatory mutations.
    • fig. S14. MFA of crtS mutants before and after acquisition of compensatory mutations.
    • fig. S15. The effect of MFA normalizations.
    • table S1. Compensatory mutations obtained after evolution of ΔcrtS mutants.
    • table S2. List of plasmids and bacterial strains.
    • table S3. Primers used in qPCR.
    • Legends for movies S1 to S4
    • References (50–64)

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    Other Supplementary Material for this manuscript includes the following:

    • movie S1 (.avi format). 3D representations of the contact map from fig. S9 (exponential growth of the WT in LB).
    • movie S2 (.avi format). 3D representations of the contact map from fig. S9 (exponential growth of the WT in MM).
    • movie S3 (.avi format). Time-lapse fluorescence microscopy of ΔcrtS filamentous cells, tagged at VC783 (Chr1) and near ori2 (Chr2), growing on an M9 MM agar pad supplemented with fructose and thiamine.
    • movie S4 (.avi format). Time-lapse fluorescence microscopy of ΔcrtS filamentous cells, tagged at VC783 (Chr1) and near ori2 (Chr2), growing on an M9 MM agar pad supplemented with fructose and thiamine.

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