Chromosomal organization and segregation in Pseudomonas aeruginosa

Abstract

The study of chromosomal organization and segregation in a handful of bacteria has revealed surprising variety in the mechanisms mediating such fundamental processes. In this study, we further emphasized this diversity by revealing an original organization of the Pseudomonas aeruginosa chromosome. We analyzed the localization of 20 chromosomal markers and several components of the replication machinery in this important opportunistic γ-proteobacteria pathogen. This technique allowed us to show that the 6.3 Mb unique circular chromosome of P. aeruginosa is globally oriented from the old pole of the cell to the division plane/new pole along the oriC-dif axis. The replication machinery is positioned at mid-cell, and the chromosomal loci from oriC to dif are moved sequentially to mid-cell prior to replication. The two chromosomal copies are subsequently segregated at their final subcellular destination in the two halves of the cell. We identified two regions in which markers localize at similar positions, suggesting a bias in the distribution of chromosomal regions in the cell. The first region encompasses 1.4 Mb surrounding oriC, where loci are positioned around the 0.2/0.8 relative cell length upon segregation. The second region contains at least 800 kb surrounding dif, where loci show an extensive colocalization step following replication. We also showed that disrupting the ParABS system is very detrimental in P. aeruginosa. Possible mechanisms responsible for the coordinated chromosomal segregation process and for the presence of large distinctive regions are discussed.

Figure 1. Progressive segregation of P. aeruginosa chromosomal loci.

(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 parS sequence positions. Four of these parS sites are clustered close to oriC, 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 x-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.

Figure 2. Position of chromosomal loci inside bacterial cells, oriented relative to the new pole of the cell.

Positions in cells smaller than 2.8 μm (A), in cells between 2.8 and 3.5 μm (B) and in cells larger than 3.5 μm (C). The y-axis represents the relative position of the focus in bacterial cells, 0 being the new pole and 1 the old pole. The markers represent the median values of relative positions, and the vertical bars represent the 25-75th percentiles of the relative positions. The positions of the single focus in one-focus cells are represented with a solid diamond and a black vertical bar, whereas the positions of the two foci in two-foci cells are indicated with an open diamond and a gray vertical bar.

Figure 3. Localization of P.aeruginosa DNA polymerase.

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.

Figure 4. Extensive chromosomal disorganization in mutants of the ParABS system.

(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 ΔparA mutant (C) and the ΔparB 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).

Figure 5. Proposed model for P. aeruginosa chromosomal organization.

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.