Long-range chromosome organization in E. coli: a site-specific system isolates the Ter macrodomain - PubMed (original) (raw)
Long-range chromosome organization in E. coli: a site-specific system isolates the Ter macrodomain
Axel Thiel et al. PLoS Genet. 2012.
Abstract
The organization of the Escherichia coli chromosome into a ring composed of four macrodomains and two less-structured regions influences the segregation of sister chromatids and the mobility of chromosomal DNA. The structuring of the terminus region (Ter) into a macrodomain relies on the interaction of the protein MatP with a 13-bp target called matS repeated 23 times in the 800-kb-long domain. Here, by using a new method that allows the transposition of any chromosomal segment at a defined position on the genetic map, we reveal a site-specific system that restricts to the Ter region a constraining process that reduces DNA mobility and delays loci segregation. Remarkably, the constraining process is regulated during the cell cycle and occurs only when the Ter MD is associated with the division machinery at mid-cell. The change of DNA properties does not rely on the presence of a trans-acting mechanism but rather involves a cis-effect acting at a long distance from the Ter region. Two specific 12-bp sequences located in the flanking Left and Right macrodomains and a newly identified protein designated YfbV conserved with MatP through evolution are required to impede the spreading of the constraining process to the rest of the chromosome. Our results unravel a site-specific system required to restrict to the Ter region the consequences of anchoring the Ter MD to the division machinery.
Conflict of interest statement
The authors have declared that no competing interests exist.
Figures
Figure 1. Chromosomal rearrangement by transposition.
The three att sites are integrated in the chromosome in the same orientation. _att_L and _att_R sites are flanked by the 5′ and 3′ parts of lacZ, respectively . An additional _att_B site (_att_B′) is inserted at a defined position. Excisive recombination promoted by Int+Xis results in the excision and circularization of the intervening segment carrying _att_P. This recombination event reconstitutes a functional lacZ. The excised molecule is not replicated and should be reintegrated for the viability of cells. Integration can occur in reconstituted _att_B (1) or in _att_B′ (2). Insertion in _att_B′ gives rise to Lac+ recombinants.
Figure 2. Travelled distance of different markers in strains with a wt or rearranged configurations.
The mean value and the standard deviation calculated for 30 independent foci are indicated by columns. The x axis represents the chromosome genetic map; the positions of the loci are in Mb from oriC. The MDs and NS regions are indicated above the graph. (A) wt strain (markers Ori-3, NSR-1, NSR-2, NSR-5, Right-2 (R2), Right-5 (R5), Ter-3, Left-1 (L1), NSL-3 and NSL-4 are indicated from left to right). (B) Strain LC13-R127-BO-NSR with a transposition of the Right MD between the Ori MD and the NSRight region (markers Ori-3, NSR-1, Right-2 (R2), Right-5 (R5), NSR-2, NSR-5, Ter-3, Left-1 (L1), NSL-3, and NSL-4 are indicated from left to right). (C) Strain LR146-R124-BL-T2 with the NSLeft region transposed between the Ter and left MDs (markers Ori-3, NSR-2, Ter-3, NSL-3, and Left-1(L1) are indicated from left to right). (D) strain LC13-R18inv/LR146-R31 with the two NS regions located on the right replication arm (markers NSR-1, NSR-2, NSL-3, NSL-4, Left-1, Right-2) (E) strain LC13-R127-BO-NSR Δ_matP_ with a transposition of the Right MD between the Ori MD and the NSRight region with a deletion of matP (markers Ori-3, NSR-1, Right-2 (R2), Right-5 (R5), NSR-2, NSR-5, Ter-3, Left-1 (L1), NSL-3 and NSL-4 are indicated from left to right).
Figure 3. Identification of insulation determinants _tid_R and _tid_L.
(A) Transposition of different fragments of Right MD between the Ori MD and the NSRight region (indicated by an arrowhead). The remaining part of the Right MD separating the NSRight region from the Ter MD is indicated by the red lines. The size of the remaining part is indicated in kb beside the name of the strain. Mobility of the marker NSR-2 is given and the presence of the insulation determinants is indicated by “+”. (B) Effect of deletions spanning the ssuEADC-ycbQRSTU region on NSR-2 mobility. The extent of the deletion is indicated below the map of the region (coordinates are indicated in bp). Mobility of the marker NSR-2 is given and the presence of the insulation determinants is indicated by “+”. (C) Effect of deletions spanning the ssuCD region on NSR-2 mobility. The extent of the deletion is indicated below the map of the region (coordinates are indicated in bp). Mobility of the marker NSR-2 is given and the presence of the insulation determinants is indicated by “+”. The position and sequence of _tid_R is indicated above the map. (D) Travelled distance of different markers in strain Δrins2 as indicated in Figure 2. The deletion of segment rins2 is schematized by a double line inside the Right MD (markers NSR-2, NSR-5, Right-2 (R2), NSL-4 are indicated from left to right). (E) Travelled distance of different markers in strain LR146-R124-BL-T with a transposition of the NSLeft region transposed inside the Left MD (markers NSR-2, NSL-3, NSL-4, and Left-1 (L1) are indicated from left to right). The insulation determinants were present in the 290 kb of the Left MD proximal to the Ter MD. (F) Travelled distance of different markers in strain LC13-R524 with an inversion between sites located in the Right and Left MD (markers NSR-2, Left-1 (L1), NSL-3, and NSL-4 are indicated from left to right). No insulation determinants were present in the 830 kb of the Left MD distal to the Ter MD; they are predicted to be present in the 135 kb segment proximal to the Ter MD and that has been positioned between the Ter and Right MDs by the inversion event.
Figure 4. _tid_R and _tid_L insulate the Ter MD.
Travelled distance of different markers in strains with a Δrins2 deletion, and carrying segment rins2.1, tid_L or a control sequence in the middle of the NSRight region. The representation is the same as in Figure 2. (A) strain Δ_rins2 ykgC-ykgD::rins2.1 with a deletion of the segment rins2 and an insertion of segment rins2.1 between ykgC and ykgD (markers NSR-2, NSR-5, Right-2 (R2), NSL-4 are indicated from left to right). (B) strain Δrins2 ykgC-ykgD::_tid_L with a deletion of the segment rins2 and insertion of _tid_L between ykgC and ykgD (markers NSR-2, NSR-5 and NSL-4 are indicated from left to right). (C) strain Δrins2 ykgC-ykgD::_tid_Rshuffled with a deletion of the segment rins2 and an insertion of shuffled sequence with a similar palindromic organization as _tid_R between ykgC and ykgD (markers NSR-2, NSR-5, NSL-4 are indicated from left to right).
Figure 5. Insulation requires the YfbV protein.
Travelled distance of different markers in strains deleted for yfbV or yfbV and matP. The representation is the same as in Figure 2. (A) strain with a deletion of yfbV (markers Ori-3, NSR-2, Right-2 (R2), Ter-3, Left-1 (L1) and NSL-4 are indicated from left to Right). (B) strain with a deletion of yfbV and matP (markers Ori-3, NSR-2, Right-2 (R2), Ter-3, Left-1 (L1) and NSL-4 are indicated from left to right).
Figure 6. The constraining process depends on MatP and ZapB.
(A–D) Travelled distance of different markers in strains deleted for zapB, deleted for segment rins2 and zapB, and in wt strain. The representation is the same as in Figure 2. (A) strain with a deletion of zapB (markers Ori-3, NSR-2, Right-2 (R2), Ter-3, Left-2 (L2), Left-1 (L1) are indicated from left to right). (B) strain with a deletion of zapB and rins2 (markers Ori-3, NSR-2, NSR-5, Right-2 (R2) are indicated from left to right). (C) small cells of wt strain before Ter MD segregation at mid-cell (markers Ori-3, NSR-5, Right-2 (R2), Ter-3 are indicated from left to right). (D) small cells of strain Δrins2 before Ter MD segregation at mid-cell (markers Ori-3, NSR-2, NSR-5, Right-2 (R2), Ter-3, NSL-4 are indicated from left to right). (E) Quantification of the co-localisation of the MatP-mCherry focus (red) with a chromosomal parS tag bound by ParB-GFP (green). The percentage of cells presenting different interfocal distances is given (left column). In the top lane, the MatP-mCherry focus co-localizes with the Ter-3 marker in the wt strain. The interfocal distance is smaller than 0.4 µm in more than 90% of the cells. In the central lane, the MatP-mCherry focus does not co-localize with the NSR-2 marker in the strain LC13-R127-BO-NSR with a transposition of the Right MD between the Ori MD and the NSRight region (see chromosome configuration in Figure 2). The interfocal distance is greater than 0.3 µm in almost 90% of the cells. In bottom lane, the MatP-mCherry focus does not co-localize with the Right-2 marker in the wt strain. The interfocal distance is greater than 0.3 µm in 90% of the cells.
Figure 7. Model for the insulation of the Ter MD by _tid_RL.
In wt cells (left panel), the Ter MD (only the Ter MD and the flanking DNA is represented) is found near the new pole. As the cell cycle progresses (see for details), the Ter MD is segregated at mid-cell. Interaction of MatP with ZapB promotes the tight association of the Ter MD with the divisome (represented by a grey oval), period indicated by the vertical black bar. At this stage, the constraining process (schematized by a wavy blue line) is apparent but does not propagate behind _tid_R and _tid_L. The constraining process would abrogate when the Ter MD dissociates from the divisome. The insulation mechanism requires a direct or indirect association of tidR with YfbV. It is not known if this association lasts during the entire cell cycle. In Δ_tid_R cells (right panel), insulation is efficient only on the left arm of the chromosome and the constraining process spreads to the right arm of the chromosome. The replication period of wt cells is represented by the green vertical bar; replication is initiated in the mother cell (indicated by a green horizontal bar) and terminates soon after birth (indicated by a horizontal red bar).
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