MukBEF activity detected along the chromosome

(A) Complementation of the ΔmukF mutant. Serial dilutions (10-1 to 10-5) of an exponential culture of MG1655 (WT), MG1655 ΔmukF ppSV38, and MG1655 ΔmukF ppSV38::mukBEF were plated on media with (Right) or without (Left) inducer (IPTG). Plates were incubated at a permissive temperature of 22°C (top) or at a non-permissive temperature of 37°C (bottom).

(B) : Percentage of anucleate cells (blue bars) in wild type (WT) and mukF mutant strains, complemented or not by the plasmid ppsV38::mukBEF, grown in minimal medium at 22°C with or without IPTG. Anucleate cells were identified and counted after DAPI staining. The histograms and error bars represent the means and standard deviations from at least three independent experiments.

(C) : Normalized Hi-C contact maps (5Kb bin resolution) obtained from a ΔmukF strain complemented with ppsV38::mukBEF after different induction times (0 min, 40 min, 2 h). Cells were grown in permissive conditions at 22°C in minimal medium. The X and Y axes represent genomic coordinates in megabases (Mb).

(D) Ratio of normalized contact maps of ΔmukF ppSV38::mukBEF grown in the presence and absence of ITPG, represented in the left panel for 40 minutes of growth. The right panel shows the ratio of normalized contact maps for 2 hours of growth with and without ITPG. A decrease or increase in contacts in the induced condition compared with the non-induced condition is represented with a blue or red color, respectively. The black line represents a schematic chromosome, with the Ter domain highlighted in light blue.

(E) Quantification of the range of cis contacts of chromosomal loci along the chromosome of a ΔmukF ppsV38::mukBEF strain grown at 22°C under four different conditions: light blue (without ITPG), red (after 20 minutes of ITPG), yellow (after 40 minutes of ITPG), and purple (after 2 hours of ITPG).

MukBEF activity initiates at different regions of the E.coli chromosome

(A) (left) Normalized 3C-seq 5kb bin contact map of strain RiTer and (right) normalized Hi-C 5Kb bin contact map of strain LiTer15. The positions of the two Ter segments are highlighted in blue on the scheme, and the dashed lines project their positions onto the matrix.

(B) Ratio of normalized contact maps of RiTer strain on RiTer ΔmukB strain (left), or RiTer on RiTer ΔmatP (middle), and LiTer15 on LiTer15 ΔmukB strain(right). A decrease or increase in contacts in the transposed cells compared to the transposed mutant cells is shown in blue or red color, respectively.

(C) The graph shows the quantification of the Hi-C diagonal width for loci along the chromosome of the RiTer strain (left panel, blue line) and the RiTer ΔmukB strain (left panel, red line), as well as LiTer15 (right panel, blue line) and LiTer15 ΔmukB strain (right panel, red line). Schematic representations of the two Ter segments are indicated below the graph, highlighting the fact that the range of contacts decreased on all Ter fragments.

The number and distribution of matS sites have different effects on MukBEF Inhibition

(A) The figure shows a schematic representation of different transpositions with the number of matS sites located on each Ter region. The three att sites are integrated into the chromosome in the same orientation, and attL/attR are fixed positions on all LiTer transpositions. attB is inserted at different positions into the Ter allowing, upon transposition, the division of the Ter domain into two subdomains, Ter1 and Ter2, containing different numbers of matS sites. The number of matS sites is indicated for the different transpositions, and the distribution of matS on the Ter2 segment is indicated in the schematic for the three transposed strains, LiTer4, LiTer7, and LiTer9.

(B) Normalized Hi-C contact map with 5Kb bin resolution of the transposed strains LiTer7, LiTer4, and LiTer4 ΔmatS28. The position of the different Ter regions is highlighted below the matrix, and by dashed lines on the matrix.

(C) Ratio of normalized 5Kb bin contact maps for the different transpositions compared to the matP mutant on the same genetic organization. The position of the different Ter regions is highlighted below the ratio, and by dashed lines on the ratio.

(D) Quantification of the Hi-C diagonal width for loci along the chromosome for the transposed strains LiTer7, LiTer4, the derivative mutant LiTer4ΔmatS28, and LiTer4ΔmatP. The schematic map below represents the LiTer7 configuration. The position of the different Ter regions is highlighted below the graph, and by dashed lines on the graph.

(E) This panel quantifies the range of contacts in the Ter2 region or in the corresponding sequence on the wild-type (WT) configuration. Boxplot representations are used, indicating the median (horizontal bar), the 25th and 75th percentiles (open box), and the rest of the population.

MukBEF preferentially binds in newly replicated regions and is exclude from Ter sequence

(A) Immunoblot analysis of mukB-flag dnaC2 strain. Exponentially growing cells were synchronized by incubating them at 40°C for 2 hours, followed by a shift to 30°C for 0, 10, 20, or 40 minutes. The replication progression was monitored by plotting the input sequence (left panel) in 50 kb bins. Normalized ChIP (ChIP over input) values for 50 kb bins are presented in the right panel, with the red asterisk indicating the peak observed in all E. coli ChIP-seq experiments. The positions of oriC and Ter are highlighted with a red dashed line and on the chromosome schematic below the figure.

(B) Immunoblot analysis was conducted on the LiTer7 mukB-flag dnaC2 strain. We observed that replication restart in this transposed strain exhibited a 20-minute lag. Therefore, cells in replication stop state were shifted to 30°C for 60 minutes to achieve a comparable replication progression to the 40-minute WT strain, as shown by the plotting of the input sequence (left panel). MukB enrichment generally followed replication progression, except in the two Ter regions, as demonstrated by the immunoblot of MukB ChIP normalized by input at 50 kb bins (right panel).

(F) Normalized Hi-C 5 kb bin contact map of ΔmukF ppsV38::mukBEF dnaC2 strain. Exponential cells were incubated at 40°C for 2 hours to prevent replication initiation (non-replicating condition) and were then shifted to 30°C for 40 minutes (right panel). MukBEF induction was performed by adding IPTG to the media for 2 hours.

identification of matS sequence and Ter domain in Gammaproteobacteria

a Ter size in kilobases. Ter is defined as the longest stretch of DNA containing matS flanked by two regions of 100 kilobases devoid of matS sites

b Number of matS sites identified in the Ter region using the MEME Suite and based on the matS consensus sequence (see Supplementary Figure 3c).

c matS density inside the Ter region calculated as the number of matS sites divided by the size of the Ter region.

d Proportion of the Ter region compared to the entire chromosome, expressed as a percentage.

Distribution of matS sites culminates at dif across γ-proteobacteria

The density of matS sequences over a 100kb-unit region was measured in 16 γ-proteobacteria, and the resulting distribution is plotted in the figure. The red line represents the 75th percentile of this distribution, while the blue line represents the 25th percentile. The light green area between these two lines represents the 50% of values closest to the median. The distribution is centered on the dif site, and the x-axis represents the genomic distance in megabases (Mb) from dif, while the y-axis represents the number of matS sequences per 100 kilobases (Kb).