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. 2016 Sep 14;12(9):e1006309.
doi: 10.1371/journal.pgen.1006309. eCollection 2016 Sep.

The MaoP/maoS Site-Specific System Organizes the Ori Region of the E. coli Chromosome into a Macrodomain

Michèle Valens  1 , Axel Thiel  1 , Frédéric Boccard  1
Affiliations

The MaoP/maoS Site-Specific System Organizes the Ori Region of the E. coli Chromosome into a Macrodomain

Michèle Valens et al. PLoS Genet. .

Abstract

The Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome. MaoP belongs to a group of proteins conserved in the Enterobacteria that coevolved with Dam methylase including SeqA, MukBEF and MatP that are all involved in the control of chromosome conformation and segregation. Analysis of DNA rings excised from the chromosome demonstrated that the single maoS site is required in cis on the chromosome to exert its effect while MaoP can act both in cis and in trans. The position of markers in the Ori region was affected by inactivating maoP. However, the MaoP/maoS system was not sufficient for positioning the Ori region at the ¼-¾ regions of the cell. We also demonstrate that the replication and the resulting expansion of bulk DNA are localized centrally in the cell. Implications of these results for chromosome positioning and segregation in E. coli are discussed.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Travelled distance of different markers in strains with WT or rearranged configurations.
Columns indicate the mean value with standard deviation calculated for 30 independent foci. The x axis represents the chromosome genetic map (position in Mb from thrABC operon). The MDs (Ori and Right) and the NSRight region are indicated above the graph: the transposed segments are indicated by a line below the WT map, the insertion point by arrowhead. (A) WT strain and its derivative upon transposition of segment ori Td-1 (coordinates 3928826–4067141; strain OriT1inRight17) in the Right MD (attB17). Markers Ori-6, Ori-7, Ori-3, Ori-4, NSR-5 and Right-5 are indicated. (B) WT strain and its derivatives upon transposition of the 318-kb long segment ori Td-4 (coordinates 4379216–55668; strain OriT4inRight17) and NSR Td-1 (strain NSRT1inRight17) in the Right MD (attB17), respectively. Markers Ori-5, Ori-3, Ori-4, NSR-1, NSR-2, NSR-5, Right-2 and Right-5 are indicated. (C) WT strain and its derivative upon transposition of 264-kb segment NSR Td-2 (coordinates 66848 to 331520; strain NSRT2inOri87.6) in the Ori MD (87.6 min). Markers Ori-3, Ori-4, NSR-1, NSR-2 and NSR-5 are indicated. (D) WT strain and its derivatives upon transposition of 154-kb segment right Td-1 (coordinates 649460–806549) at two different positions, 92.7 min (position 4302204; strain RightT1in Ori92.7) and 87.6 min (position 4067141; strain RightT1inOri87.7), respectively. Markers Ori-1, Ori-3, Ori-4, Right-2 and Right-5 are indicated.
Fig 2
Fig 2. Mapping of determinants required for Ori MD structuring.
(A) Travelled distance of different markers in strains with a WT or rearranged configurations carrying a segment right Td-2 inserted at various positions: 4067141 (a), 4024867 (b), 3998022 (c), 3947900 (d) and 3928826 (e) in the Ori MD. The representation is the same as in Fig 1. Markers Ori-3, Ori-4 and Ori-5 are indicated. A reference dashed red line indicates the average mobility of markers Ori-3, Ori-4 and Ori-5 in a wt context. (B) Effect of deletions spanning the 69 kb region (from coordinates 3928900–3998000) on Ori-3 and Ori-4 mobility. The extent of the region remaining upon the deletion is indicated. Deletion of the leftmost 10 kb (coordinates 3929000–3939000, from gene rbsD to yieP), of the 10 kb region between ilvL and ppiC (coordinates 3948000–3957000) and of the 16 kb region from wzzE to aslB (coordinates 3967000–3985000) had no strong effects on the mobility of markers Ori-3 and Ori-4; a strong increase in mobility of Ori-3 and Ori-4 was observed when the 2 kb region encompassing gene hdfR, yifE and yifB (coordinates 3944500–3946500) was deleted. Mobility of markers Ori-3 and Ori4 are indicated; the loss of constraints are indicated by “-”and DNA constraint indicated by “+”. (C) Effect of deletions spanning the hdfR-yifE-yifB region (from coordinates 3944752–3948043) on Ori-3, Ori-4 and NSR-2 mobility. The extent of the region remaining upon the deletion is indicated. Mobility of markers Ori-3, Ori4 and NSR-2 are indicated; the absence of constraint is indicated by “-”and DNA constraint indicated by “+”.
Fig 3
Fig 3. Identification of determinants required for Ori MD structuring.
(A) Effect of the presence of maoP in trans or the insertion of maoS at an ectopic position on deletions spanning the hdfR-yifE region (from coordinates 3944752–3948043) on Ori-3, Ori-4 and NSR-2 mobility. The extent of the region remaining upon the deletion is indicated. Mobility of markers Ori-3, Ori4 and NSR-3 are indicated; the loss of DNA constraint is indicated by “-” and DNA constraint indicated by “+”. (B) Programmed excision of chromosomal segments by site-specific recombination. The two att sites are integrated in the chromosome in the same orientation. Excisive recombination promoted by Int+Xis results in the excision and circularization of the intervening segment carrying attP or attB (depending on the order of attL and attR sites in the chromosome). attL and attR sites are flanked by the 5’ and 3’ parts of lacZ, respectively. The excised segment is devoid of replication origin and is not replicated. Integrative recombination between attP and attB occurs at very low frequency preventing fusion of the two molecules. (C) Travelled distance of Ori markers upon excision of different segment in the Ori region. The ori Td-3 segment carries maoS and maoP indicated by a red square. Mobility of markers Ori-3, Ori4 or Ori-6 are indicated; the loss of constraints is indicated by “-”and DNA constraint indicated by “+”.
Fig 4
Fig 4. Effect of the inactivation of the MaoP-maoS system.
(A) Travelled distance of different markers in WT strain and a maoP deletion derivative. Columns indicate the mean value with standard deviation calculated for 30 independent foci. The x axis represents the chromosomal genetic map (position in Mb from thrABC operon). The MDs (Ori, Right, Left and Ter) and the NS regions are indicated above the graph. (B) Colonies of strains carrying a normal (white colonies) or an inverted configuration (blue colonies) upon DNA inversion between Ori and Right MDs in a WT and maoP genetic background. (C) Positioning of chromosomal marker Ori-3 in a WT (left panel) and in a maoP mutant (right panel) observed in 400 cells. Cells are sorted for length, ascending from top to bottom. In the heat maps blue corresponds to low and red to high intensity. The diagram represents the relative position of the foci as a function of the cell size.
Fig 5
Fig 5. Exclusion from the nucleoid of large DNA rings looped out from the Ori region of the chromosome.
Montage of merged pictures of parSP1 (green), DAPI staining (red) and phase-contrast micrographs (grey) of FBG150 cells upon excision of chromosomal DNA segments carrying the parS tag. The extent of the excised segment is indicated on the diagram above the micrographs (segments a, b and c). Control sample in the absence of excision is presented on the top lane (no excision). Black arrowheads indicate Ori markers present on excised rings. Scale bar indicate 2 μm.
Fig 6
Fig 6. E. coli nucleoid positioning and chromosome segregation.
(A) The extent of the excised segment is indicated on the diagram below the map of the NSL-Ori region (segments a, b and c). (B) Montage of merged pictures of parSP1 (green), DAPI staining (red) and phase-contrast micrographs (grey) of MG1655 cells upon excision of chromosomal DNA segments a, b and c carrying oriC and a parS (Ori-5 or Ori-6). Control sample in the absence of excision is presented on the top panels. (C) Montage of merged pictures of MG1655 cells upon excision of chromosomal DNA segments carrying oriC and the parS tag (Ori-5 and Ori-6; indicated in green), together with a parST1 (indicated in yellow) on the remnant part either in Ori region (panel a) or in Ter region (panel b) or expressing a MatP-mCherry fusion protein (panel c), HU-mCherry staining (red; panel a) and phase-contrast micrographs (grey). Control sample in the absence of excision is presented on the top panels. (D) Montage of merged pictures of parSP1 Ori-6 (green), DAPI staining (red) and phase-contrast micrographs (grey) of MG1655 cells upon excision of chromosomal DNA segment b carrying oriC and the parS tag (Ori-6) in the presence of cephalexin. Control sample in the absence of excision is presented. Black arrowheads indicate excised rings carrying Ori markers. (E) Time-lapse experiment representing the dynamics of parSP1 Ori-6 tag upon excision of segment b. Montage of merged pictures of MG1655 cells upon excision of chromosomal DNA segments carrying oriC and the parS tag (Ori-6) and expressing HU-mCherry. Positioning of the focus was observed for 300 min with 10 min intervals. Black arrowheads indicate chromosome remnants. Scale bar indicate 2 μm.
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This work was supported by the Agence Nationale de la Recherche grant ANR-12-BSV8-0020-01 to FB and by CNRS. The "Agence Nationale de la Recherche and the CNRS had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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