Effect of Right and Left transposition on long distance DNA interactions.

<p>Histograms of recombination frequency between <i>attL</i> and <i>attR</i> sequences in WT, RT, and LT strains. The y-axis indicates the percentage of recombination between <i>attL</i> and <i>attR</i> sequences, obtained as described in Materials and Methods with 20’ induction at 36°C. The relative position of each <i>att</i> sequence used in the experiment is represented on the MD map on top of each panel. Histograms show the average of at least 3 independent experiments with their respective standard-deviation. Frequencies obtained in the WT strain are represented with the black bars, in the RT strain (A,C) or LT strain (B,D) with the gray bars and in the Δ<i>matP</i> RT strain (C), or Δ<i>matP</i> LT strain (D) with the light gray bars. * indicates that no <i>att</i>L<i>-att</i>R recombinant was obtained.</p

Chromosomal rearrangement by transposition.

<p>Chromosomal map of the 4 MDs (Ori: green, Right: red, Ter: light blue, Left: Dark blue) and the two non-structured regions (dashed black line) in the WT strain, the Right transposed strain and the Left transposed strain. <i>att</i> sequences used for the RT transposition are indicated by the green barre, the Right fragment between <i>attR127</i> (651 kb) and <i>attLlc13</i> (1099 kb)(dashed red line) was transposed at the position <i>attB O-NSR</i> (153 kb). <i>att</i> sequences used for the LT transposition are marked in blue, the NSL fragment between <i>attR124</i> (2892 kb) and <i>attL146</i> (3697 kb, dashed blue line) was transposed at the position <i>attB LT2</i> (1920 kb).</p

The position of the replication origin changes the Right and Left boundaries.

<p>Histograms of recombination frequencies between <i>att</i>L and <i>att</i>R sequences in WT, and Inv strain. Recombination frequencies between different <i>attR/L</i> sites are indicated on the y-axis. These values are the average of at least 3 independent experiments and the error bars correspond to the standard deviation. For both panels, strains were grown in minimal medium and the recombinase production was obtained by shifting cultures at 38°C for 10’. Relative position of each <i>att</i> sequence used in the experiment is represented on the MD map on top of each panel. The name of the <i>att</i> sequence, and the MD which they belong to, are indicated on the x-axis. The genetic backgrounds are indicated below the histogram (A), or with a color code (B). * indicates that the recombination frequency obtained was repeatedly 0.</p

The interaction limit of the Ori MD.

<p>Graphical representation of the Ori MD (green box), NSR region (gray line) and Right MD (red box). Coordinates of the <i>attR/L</i> sequences are indicated in fonction of their distance from the zero pb reference of the MG1655 genome for both configuration WT and RT. Percentages of recombination between <i>attL</i> and <i>attR</i> obtained after induction of 20’ at 36°C or 10’ at 37°C are shown. The histograms correspond to an average of at least 3 independent experiments with standard-deviations.</p

Position of chromosomal loci in the Ori and Right MD depending of <i>oriC</i> position.

<p>MD map of the <i>E</i>. <i>coli</i> chromosome are represented with the position of the Ori-4, Right-2, FROS-Tag in an Inv (A), or <i>oriCZ</i> (B) configurations. For each panel, the position of foci in cell containing 2 foci (cell with one focus are show in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006758#pgen.1006758.s004" target="_blank">S4 Fig</a>), are represent in function of the long axis of the cell (y-axis) and in function of the cell length (x-axis). Red dots and bars correspond to the localization of the Right-2 loci and green ones to the Ori-4 loci. (C) Histogram of the percentage of the population presenting different interfocal distance (<0.2 to > 0.5) between Ori-4 and Right-2 in the WT strain (blue), Inv strain (red) and <i>oriCZ</i> strain (green). Numbers on the first columns show the percentage of cells where both foci are co-localized.</p

Position of chromosomal loci in the Ori, NSR region and Right MD.

<p>MD maps of the <i>E</i>. <i>coli</i> chromosome are represented with the position of the Ori-4, Right-2, NSR-5 FROS-Tag in a WT strain. For each panel, the position of foci in cell containing 2 foci, are represented in function of the long axis of the cell (y-axis) and in function of the cell length (x-axis). Green dots and bars show the position of the Ori-4 loci and red dots and bars correspond to the localization of the Right-2 (A) or NSR-5(B) loci. (C) Histograms of the percentage of the population presenting different interfocal distance (<0.2 to > 0.5) between Ori-4 and Right-2 (dark blue) or Ori-4 and NSR-5 (light blue).</p

SOS induction in Δ<i>holD</i> and Δ<i>holD</i> suppressed mutants.

<p>N indicates the number of independent experiments. Since JJC2067 isolated colonies could not be used, JJC2067 and JJC2068 overnight cultures propagated in MM lacking IPTG were diluted 50 fold and grown in MM for the experiment.</p><p>SOS induction in Δ<i>holD</i> and Δ<i>holD</i> suppressed mutants.</p

pGB-<i>dinB</i> is lethal to Δ<i>holD argE::ssb</i> only when the SOS response is induced.

<p>Plain, dotted and hatched boxes correspond to pGB2, pGB-<i>dinB</i> and pGB-<i>dinB</i>ΔC5 transformation efficiencies, respectively, when grown on MM after overnight incubation at 37°C, normalized to pGB2 transformation efficiency on LB at 37°C in the same strain (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004719#pgen.1004719.s008" target="_blank">Table S2</a> for transformation efficiencies on LB). Wild-type (wt) JJC40 and JJC1945; Δ<i>holD argE::ssb lexAind</i>, JJC6133; Δ<i>holD argE::ssb</i>, JJC6110, <i>lexA</i>Def JJC6488. Averages of three to four experiments are shown. pGB-<i>dinB</i> transformants appeared on MM at 37°C in two days in one JJC6110 (Δ<i>holD argE::ssb</i>) experiment out of four, and in the same way as clones obtained on LB (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004719#pgen.1004719.s005" target="_blank">Figure S5</a>), they could not be propagated.</p

In an AB1157 background, a <i>ssb</i> gene duplication is sufficient to suppress Δ<i>holD</i> mutant growth defects.

<p>Serial dilutions of suspended colonies were spotted on MM and incubated as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004719#pgen-1004719-g001" target="_blank">Figure 1</a> legend. Top panel: Wild-type, JJC2069; <i>argE::ssb</i>, JJC6047; Δ<i>holD</i>, JJC6050 cured of pAM-<i>holD</i>; Δ<i>holD argE::ssb</i>, JJC6110; Δ<i>holD lexAind</i>, JJC1524 cured of pAM-<i>holD</i>; Δ<i>holD lexAind argE::ssb</i>, JJC6077 cured of pAM-<i>holD</i>. Bottom panels: MG1655, JJC3523; MG1655 Δ<i>holD</i>, JJC6363 cured of pAM-<i>holD</i>; MG1655 Δ<i>holD lexAind</i>, JJC6420 cured of pAM-<i>holD</i>; MG1655 Δ<i>holD argE::ssb</i>, JJC6394 cured of pAM-<i>holD</i>; MG1655 Δ<i>holD argE::ssb lexAind</i>, JJC 6419 (spontaneously cured of pAM-<i>holD</i> during construction at 30°C).</p

Duplicated sequence in JJC2394.

<p>Schematic representation of several JJC2394 sequence reads aligned to the wild-type <i>E.coli</i> genome using GenomeStudio Software (Illumina). The black box corresponds to a duplication of the 10 kb chromosome region shown below it. A 6 bp repeated sequence was identified at the duplication boundaries.</p