Regional Control of Chromosome Segregation in Pseudomonas aeruginosa

Abstract

Chromosome segregation in bacteria occurs concomitantly with DNA replication, and the duplicated regions containing the replication origin oriC are generally the first to separate and migrate to their final specific location inside the cell. In numerous bacterial species, a three-component partition machinery called the ParABS system is crucial for chromosome segregation. This is the case in the gammaproteobacterium Pseudomonas aeruginosa, where impairing the ParABS system is very detrimental for growth, as it increases the generation time and leads to the formation of anucleate cells and to oriC mispositioning inside the cell. In this study, we investigate in vivo the ParABS system in P. aeruginosa. Using chromatin immuno-precipitation coupled with high throughput sequencing, we show that ParB binds to four parS site located within 15 kb of oriC in vivo, and that this binding promotes the formation of a high order nucleoprotein complex. We show that one parS site is enough to prevent anucleate cell formation, therefore for correct chromosome segregation. By displacing the parS site from its native position on the chromosome, we demonstrate that parS is the first chromosomal locus to be separated upon DNA replication, which indicates that it is the site of force exertion of the segregation process. We identify a region of approximatively 650 kb surrounding oriC in which the parS site must be positioned for chromosome segregation to proceed correctly, and we called it "competence zone" of the parS site. Mutant strains that have undergone specific genetic rearrangements allow us to propose that the distance between oriC and parS defines this "competence zone". Implications for the control of chromosome segregation in P. aeruginosa are discussed.

Fig 1

Chromatin Immunoprecipitation of ParB-3xFLAG, in the wild type background (strain IVGB379) (A), in the ΔparS123 background (strain VLB3) (B) and in the ΔparS1234 background (strain VLB4) (C). Enrichment folds between the immunoprecipitated (IP) and the input (IN) fractions are represented for each base of the genome, according to its distance from oriC. The insets represent a zoom of the region containing the four parS sites bound by ParB. Dashed vertical lines represent the position of the proposed parS sites from [21]. Red italicized letters indicated ParB accessory binding sites, and blue italicized letters represents the promoter region of dnaA. Grey stars indicate peaks that are less prominent but still found in the different genetic backgrounds. The red dotted lines indicate the significant enrichment. Precise description of the different peaks is given in S1 Table.

Fig 2. Impact of parS site location on the chromosome on generation time and amount of anucleate cell found in liquid cultures.

(A) Schematic representation of the position of the parS sites introduced in the ΔparS1234 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 (ΔparS123), VLB1 (ΔparS1234), VLB63 (ΔparS1234 parS2 +6.5), VLB62 (ΔparS1234 parS9 +6.5), VLB69 (ΔparS1234 parS -898), VLB70 (ΔparS1234 parS -440), IVGB481 (ΔparS1234 parS -330), VLB66 (ΔparS1234 parS +347), IVGB479 (ΔparS1234 parS +449), VLB73 (ΔparS1234 parS +545) and IVGB480 (ΔparS1234 parS +552)were used.

Fig 3. ParB binding to parS in the presence of ParA induces its positioning to the 0.2/0.8 relative cell length.

The pPSV38-NGFP-ParB plasmid was introduced in different strains, and NGFP-ParB localization was observed in cells grown in minimal medium supplemented with citrate. Foci numbers for each strain are indicated in S2 Table. (A) represents the localization of the 2 foci in cells (randomly oriented) containing two foci, for each genetic background, whereas (B) represents the distance between the 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×IQR or more above the 75^th percentile or 1.5×IQR or more below the first 25th percentile quartile. Representative images are shown in (C). Scale bar is indicated. Raw data are presented in S5 Fig. Strains IVGB469 (ΔparS123), VLB66 (ΔparS1234 parS +347), IVGB480 (ΔparS1234 parS +552), VLB69 (ΔparS1234 parS -898) and IVGB317 (ΔparA) were used.

Fig 4. Positioning inside the cells of chromosomal loci located close to an ectopic parS site.

(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×IQR or more above the 75^th percentile or 1.5×IQR 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 oriC (strains IVGB492 and VLB21); 327-R, +327 kb from oriC (strain IVGB478); 628-R, +628 kb from oriC (strains VLB23 and VLB140); 851-L, -851 kb from oriC (strains IVGB509 and IVGB510).

Fig 5. The parS site is the site of force exertion of the segregation process.

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 parS. Numbers of cell considered are shown below. Schematics of the different chromosomal configurations are represented; the position of the parS site is indicated in grey, the position of oriC 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 (ΔparS1234 parS +347 background), VLB140 (ΔparS1234 parS +552 background) and IVGB510 (ΔparS1234 parS -898 background).

Fig 6. Identification of the “competence zone” of P. aeruginosa parS site.

Log2 of ratio of insertion numbers (normalized to the total number of reads for each experiment) of a mariner transposon containing a parS site and a standard mariner transposon in the ΔparS1234 mutant, calculated for 10 kb windows, and represented according to the distance from oriC. The black dashed line represents the position of oriC, the grey dashed lines the position of the ribosomal operons, and the red dashed lines the position of the parS sites introduced in the ΔparS1234 mutant presented in this study (Fig 2).

Fig 7. Impact of rrnD deletion on the amount of anucleate cells found in liquid culture for strains containing a parS site located 330 and 898 kb on the left of oriC (ΔparS1234 parS -330 (strains IVGB481 and IVGB524) and ΔparS1234 parS -898 (strains VLB69 and IVGB526), respectively).

A schematic representation of the positioning of parS and rrnD 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 t test. *, P<0.001.

Fig 8. Genetic analysis of the parS “competence zone”.

Generation of programmed chromosome rearrangements (using the lambda derived recombination system) bringing part of the “Competence zone” closer to a parS site located 851 kb on the left of oriC (A) or 628 kb on the right of oriC (B). Schematic representations of the inverted regions, as well as the position on the chromosome of the parS, attL and attR sites, oriC and rrn 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 t test. *, P<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).