Progressive segregation of <i>P. aeruginosa</i> chromosomal loci.

<p>(A) Position of each chromosomal locus on the PAO1 chromosomal map. Each locus is represented by a colored tag on the PAO1 chromosome according to its position. The color code is used for all figures. Black tags indicate rRNA operons. Grey tags represent <i>parS</i> sequence positions. Four of these <i>parS</i> sites are clustered close to <i>oriC</i>, four more are dispersed between positions 851-L and 628-R, and two are localized in the “right” replichore. (B) Average percentage of one-focus cells (solid diamonds) and two-foci cells (open diamonds) in a bacterial population grown in minimal medium supplemented with citrate. Cells with no visible focus were removed from this analysis (they constituted between 5 to 10% of the cells). The proportion of cells exhibiting more than two foci was always smaller than 0.5 %. The <i>x</i>-axis represents the positions of the loci on the chromosomal map. Values from four to eleven experiments were averaged, and the error bars represent standard deviations. (C) The average percentage of two-foci cells in a bacterial population grown in minimal medium supplemented with citrate, according to cell size, for each chromosomal locus of the “right” replichore (left panel) or of the “left” replichore (right panel). Values from four to eleven experiments were averaged, and the error bars represent standard deviations.</p

Localization of <i>P.aeruginosa</i> DNA polymerase.

<p>EGFP-labeled replisome protein DnaX was observed in minimal medium supplemented with citrate (A) or glucose and casamino acids (B). For each panel, the upper left area shows a sample of representative cells; the lower left area presents the amount of cells exhibiting zero (white), one (blue), two (red) or three (green) fluorescent foci according to cell size. The upper right area presents the relative positions of the focus in one-focus cells, and the lower left area presents the relative positions of the foci in two-foci cells.</p

Extensive chromosomal disorganization in mutants of the ParABS system.

<p>(A) Percentages of the population presenting a given number of foci corresponding to the Ter locus or the Ori locus. Positioning of chromosomal loci located in the Ori region (left panels) and in the Ter region (right panels) in a wild type PAO1 strain (B), the Δ<i>parA</i> mutant (C) and the Δ<i>parB</i> mutant (D) grown in minimal medium supplemented with citrate. The position of the foci in cells containing 1 (upper panels) or 2 (bottom panels) foci are presented. Ori locus: 82-R in (A), (B) and (C). Ter loci: 2,957-R in (A), 3,090-L in (B) and (C).</p

Proposed model for <i>P. aeruginosa</i> chromosomal organization.

<p>Organization in minimal medium supplemented with citrate (A) or glucose and casamino acids (B). Black lines represent fully replicated chromosomes, whereas grey lines represent partially replicated chromosomes. Colored markers represent chromosomal loci, and yellow diamonds represent replisomes.</p

Impact of <i>parS</i> site location on the chromosome on generation time and amount of anucleate cell found in liquid cultures.

<p>(A) Schematic representation of the position of the <i>parS</i> sites introduced in the Δ<i>parS1234</i> mutant. (B) Generation times (white bars, scale on the left axis) and percentage of anucleate cell (grey bars, scale on the right axis) of the different strains used in this study. Histograms and error bars represent the mean and standard deviation for at least three independent experiments. Strains IVGB469 (Δ<i>parS123</i>), VLB1 (Δ<i>parS1234</i>), VLB63 (Δ<i>parS1234 parS2</i> +6.5), VLB62 (Δ<i>parS1234 parS9</i> +6.5), VLB69 (Δ<i>parS1234 parS</i> -898), VLB70 (Δ<i>parS1234 parS</i> -440), IVGB481 (Δ<i>parS1234 parS</i> -330), VLB66 (Δ<i>parS1234 parS</i> +347), IVGB479 (Δ<i>parS1234 parS</i> +449), VLB73 (Δ<i>parS1234 parS</i> +545) and IVGB480 (Δ<i>parS1234 parS</i> +552)were used.</p

The <i>parS</i> site is the site of force exertion of the segregation process.

<p>Fractions of cells presenting 3 foci for two chromosomal tags in different genetics background (shown in top) are indicated in the wild type strain, and in a strain with an ectopic <i>parS</i>. Numbers of cell considered are shown below. Schematics of the different chromosomal configurations are represented; the position of the <i>parS</i> site is indicated in grey, the position of <i>oriC</i> in black, and chromosomal loci in blue and red. Strains IVGB292, IVGB168 and IVGB173 were used for the wild type background, as well as strains IVGB478 (Δ<i>parS1234 parS</i> +347 background), VLB140 (Δ<i>parS1234 parS</i> +552 background) and IVGB510 (Δ<i>parS1234 parS</i> -898 background).</p

Impact of <i>rrnD</i> deletion on the amount of anucleate cells found in liquid culture for strains containing a <i>parS</i> site located 330 and 898 kb on the left of <i>oriC</i> (Δ<i>parS1234 parS</i> -330 (strains IVGB481 and IVGB524) and Δ<i>parS1234 parS</i> -898 (strains VLB69 and IVGB526), respectively).

<p>A schematic representation of the positioning of <i>parS</i> and <i>rrnD</i> on the chromosome is shown on the left. Histograms and error bars represent the mean and standard deviation for at least three independent experiments. Significant differences between strains were determined by <i>t</i> test. *, <i>P</i><0.001.</p

Genetic analysis of the <i>parS</i> “competence zone”.

<p>Generation of programmed chromosome rearrangements (using the lambda derived recombination system) bringing part of the “Competence zone” closer to a <i>parS</i> site located 851 kb on the left of <i>oriC</i> <b>(A)</b> or 628 kb on the right of <i>oriC</i> <b>(B)</b>. Schematic representations of the inverted regions, as well as the position on the chromosome of the <i>parS</i>, <i>attL</i> and <i>attR</i> sites, <i>oriC</i> and <i>rrn</i> operons are shown on the left. Impact of these inversions on the amount of anucleate cells is shown on the right. Histograms and error bars represent the mean and standard deviation for at least three independent experiments. Significant differences between strains were determined by <i>t</i> test. *, <i>P</i><0.001. Strains VLB271, VLB272, VLB 273, VLB276, VLB277 and VLB278 were used in (A), and strains IVGB556, IVGB557, IVGB558, VLB333, VLB334 and VLB335 were used in (B).</p

Identification of the “competence zone” of <i>P</i>. <i>aeruginosa parS</i> site.

<p>Log2 of ratio of insertion numbers (normalized to the total number of reads for each experiment) of a <i>mariner</i> transposon containing a <i>parS</i> site and a standard <i>mariner</i> transposon in the Δ<i>parS1234</i> mutant, calculated for 10 kb windows, and represented according to the distance from <i>oriC</i>. The black dashed line represents the position of <i>oriC</i>, the grey dashed lines the position of the ribosomal operons, and the red dashed lines the position of the <i>parS</i> sites introduced in the Δ<i>parS1234</i> mutant presented in this study (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006428#pgen.1006428.g002" target="_blank">Fig 2</a>).</p

Positioning inside the cells of chromosomal loci located close to an ectopic <i>parS</i> site.

<p>(A) represents the localization of the 2 foci in cells (randomly oriented) containing two foci, for each genetic background, whereas (B) represents the interfocal distance between these two foci. Boxplot representations are used, indicating the median (horizontal bar), the 25th and the 75^th percentile (open box) and the rest of the population except for the outliers (whiskers). Outliers are defined as 1.5×<i>IQR</i> or more above the 75^th percentile or 1.5×<i>IQR</i> or more below the first 25th percentile quartile. Genetic background in which each locus is observed are indicated, as well as the number of cells considered. Positions of the chromosomal tags: 82-R, +82 kb from <i>oriC</i> (strains IVGB492 and VLB21); 327-R, +327 kb from <i>oriC</i> (strain IVGB478); 628-R, +628 kb from <i>oriC</i> (strains VLB23 and VLB140); 851-L, -851 kb from <i>oriC</i> (strains IVGB509 and IVGB510).</p