FtsK actively segregates sister chromosomes in Escherichia coli - PubMed (original) (raw)

FtsK actively segregates sister chromosomes in Escherichia coli

Mathieu Stouf et al. Proc Natl Acad Sci U S A. 2013.

Abstract

Bacteria use the replication origin-to-terminus polarity of their circular chromosomes to control DNA transactions during the cell cycle. Segregation starts by active migration of the region of origin followed by progressive movement of the rest of the chromosomes. The last steps of segregation have been studied extensively in the case of dimeric sister chromosomes and when chromosome organization is impaired by mutations. In these special cases, the divisome-associated DNA translocase FtsK is required. FtsK pumps chromosomes toward the dif chromosome dimer resolution site using polarity of the FtsK-orienting polar sequence (KOPS) DNA motifs. Assays based on monitoring dif recombination have suggested that FtsK acts only in these special cases and does not act on monomeric chromosomes. Using a two-color system to visualize pairs of chromosome loci in living cells, we show that the spatial resolution of sister loci is accurately ordered from the point of origin to the dif site. Furthermore, ordered segregation in a region ∼200 kb long surrounding dif depended on the oriented translocation activity of FtsK but not on the formation of dimers or their resolution. FtsK-mediated segregation required the MatP protein, which delays segregation of the dif-surrounding region until cell division. We conclude that FtsK segregates the terminus region of sister chromosomes whether they are monomeric or dimeric and does so in an accurate and ordered manner. Our data are consistent with a model in which FtsK acts to release the MatP-mediated cohesion and/or interaction with the division apparatus of the terminus region in a KOPS-oriented manner.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Systems used for visualization of chromosome loci. (A) Relevant map of constructs for fluorescent protein production. Plasmid pMS11 is a pSC101 derivative carrying a lacZp-mCherry-Δ30ParB P1 construct. GFP-Δ23ParBpMT1 and TetR-GFP were produced from chromosome-borne constructs at the lac and ara loci, respectively. (B) Examples of micrographs showing foci of the indicated fluorescent proteins in strains containing loci tagged with the relevant binding sites (ydgJ::tetO or parS P1; ydcB::parS P1, and ydeU::parS pMT1). (C) The percentage of cells with a single focus or two foci of the ydgJ (ter) or ydhJ (right) loci when visualized using fluorescent proteins produced from constructs shown in A.

Fig. 2.

Fig. 2.

The _ori_-to-ter segregation of the chromosome. The ydeU locus was tagged using the ParBpMT1 system (red arrowhead),and other loci, indicated inside the chromosome maps, were tagged with the ParBP1 system (green arrowheads). Positions of loci on the chromosome are indicated by lines representing ori (top) and dif (bottom). The number of cells analyzed is given (n). Cells were classified by the number of foci of each locus (shown in cartoons on the _x_-axis; the empty cell indicates cells that fall in none of the first four categories). Bars show the mean percentage of each category in the population (_y_-axis) with individual measured ranges. Data reflect at least two independent experiments.

Fig. 3.

Fig. 3.

ter segregation is sequential and ends at dif. (A) Map of the loci used, with coordinates indicated. The black and white box represents the dif site. Red loci were tagged with both ParB-derived systems, black loci were tagged with the TetR-derived system, and the ydgJ and ycgY loci were tagged with all three systems. (B) Intracellular position of foci tagged with parS P1 from their farthest pole (_x_-axis) as a function of cell length (_y_-axis). (Left) Cells with a single focus (black dots). (Right) Cells with two foci (red and green dots). Loci are indicated, as well as the number of cells analyzed and the mean interfocal distance (IFd). (C) Plot of the interfocal distance in cells with two foci (black curve and dots, left axis) and the mean number of foci per cell (blue curve and squares, right axis) as a function of the distance from dif (kb, _x_-axis). (D) Number of foci of the indicated loci in different cell categories. Strains carried an SSB-mCherry fusion. Postreplicative cells are >3 μm long and harbor no SSB focus and no constricting septum. Dividing cells have a constricting septum. (E) Tagged loci are indicated with their position relative to dif. Red arrowheads indicate a parS pMT1 tag, and green arrowheads indicate a parS P1 tag. Cells were classified by the number of foci of each locus (shown in cartoons on the _x_-axis; the empty cell indicate cells that fall in none of the first four categories). Bars show the mean percentage of each category in the population (_y_-axis) with individual measured ranges. Data reflect at least two independent experiments and more than 600 cells.

Fig. 4.

Fig. 4.

Segregation of ter in _recA_− strains. (A_–_C) Tagged loci are indicated in their position relative to dif (the black and white box). Red arrowheads indicate a parS pMT1 tag, and green arrowheads indicate a parS P1 tag. Cells were classified by the number of foci of each locus (shown in cartoons on the _x_-axis; the empty cell indicate cells that fall in none of the first four categories; blue bars indicate dead cells, see also

Fig. S6

). Bars show the mean percentage of each category in the population (_y_-axis) with individual measured ranges. Data reflect at least two independent experiments and 600 cells.

Fig. 5.

Fig. 5.

Segregation of ter in ftsK mutant strains. Tagged loci are indicated in their position relative to dif (the black and white box). Red arrowheads indicate a parS pMT1 tag, and green arrowheads indicate a parS P1 tag. (A) ftsK _ATP_- strain. (B_–_F) ftsK KOPSblind strains. Cells were classified by the number of foci of each locus (shown in cartoons on the _x_-axis; the empty cell indicates cells that fall in none of the first four categories; blue bars indicate dead cells; see also

Fig. S6

). Bars show the mean percentage of each category in the population (_y_-axis) with individual measured ranges. Data reflect at least two independent experiments and 600 cells.

Fig. 6.

Fig. 6.

Segregation of ter in _matP_− strains. (A_–_C) Tagged loci are indicated in their position relative to dif (the black and white box). Red arrowheads indicate a parS pMT1 tag, and green arrowheads indicate a parS P1 tag. Cells were classified by the number of foci of each locus (shown in cartoons on the _x_-axis; the empty cell indicates cells that fall in none of the first four categories). Bars show the mean percentage of each category in the population (_y_-axis) with individual measured ranges. Data reflect at least two independent experiments and 600 cells.

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