ParB phylogeny and <i>parS</i> sequences of the Par systems used in this study.

<p>ParB Phylogeny was obtained using the multiple sequence alignment webPRANK of the European Bioinformatic Institute (<a href="http://www.ebi.ac.uk/goldman-srv/webprank/" target="_blank">http://www.ebi.ac.uk/goldman-srv/webprank/</a>). The Par systems are from the following replicons: c1, chromosome 1 of <i>Burkholderia cenocepacia</i> J2315; G4, plasmid 2 of <i>Burkholderia vietnamiensis</i> G4; 12D, plasmid 1 of <i>Ralstonia pickettii</i> 12D; c3, chromosome 3 of <i>B</i>. <i>cenocepacia</i> J2315; 12J, plasmid integrated in the chromosome of <i>Ralstonia pickettii</i> 12J; pBC, plasmid pBC of <i>B</i>. <i>cenocepacia</i> J2315. Families correspond to those previously established based on a wider collection of Par systems. All <i>parS</i> sequences form a perfect palindrome (indicated by the inverted arrows with specific colours for specific sequences), except <i>parS</i>12J which displays non complementary bases (boldface).</p

Binding scores for palindromic <i>parS</i> and derivatives.

<p>(A) ParB c1 <i>B</i>. <i>cenocepacia;</i> (B) ParB c3 <i>B</i>. <i>cenocepacia;</i> (C) ParB plasmid pBC <i>B</i>. <i>cenocepacia</i>; (D) ParB plasmid 1 <i>Ralstonia picketti</i> 12D; (E) ParB plasmid 2 <i>Burkholderia vietnamiensis</i> G4. Binding scores correspond to 100-fold the VR value at the end of the SPRi injection. The average scores for the wild-type (wt) and random (rdm) sequence are represented as lines, green and red respectively. Average scores for the half-<i>parS</i> sequences are shown as orange lines. For double symmetric substitutions, the score is directly pointed on the graph as the base substituting the wild-type one indicated below on the x-axis. Changes are indicated for one arm only but correspond to symmetrical changes (<i>e</i>.<i>g</i> the change T1C in <i>parS</i>c1 means T1C and A16G). For each change statistical analysis of the BS were performed; the substitutions displaying a significantly different BS relative to that of non specific DNA are wrote in red.</p

Binding scores of ParB12 J for <i>parS</i>c3 non-<i>parS</i>c3 sequences and derivatives.

<p>Blue lines correspond to the BS of the palindromic sequence <i>parS</i>c3 (A) and non <i>parS</i>c3 (B). Legend is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177056#pone.0177056.g003" target="_blank">Fig 3</a>.</p

Experimental design.

<p><b>(A)</b> Structures of the 5’-thiol-labelled 57-mer single strand oligonucleotides carrying carrying <i>parS</i>, exemplified for <i>parS</i>c1. The <i>parS</i> sequence forms by the annealing of two inverted repeats of 26 bases (arrowed boxes). The upper structure shows the wild-type (wt) 16 bp palindrome of <i>parS</i>c1 by two arrowed black lines and with bp positions numbered. Three sequences modified from <i>parS</i>c1 are indicated below: randomly modified (rdm), one arm modified (half), and the double symmetric change T1C-A16G, (changed sequence is written in red) <b>(B)</b> Typical SPRi image captured at the end of injection (240 sec) of 10μg/ml of crude extract enriched in ParB c1. Each DNA probes was distributed in duplicate in two different localizations, as shown by white rectangles with <i>parS</i> wild type sequence (c1 wt) and white dotted rectangles with randomized sequence (c1 rdm). <b>(C)</b> Examples of kinetics curves for the six ParB-<i>parS</i> systems. For each ParB, the reflectivity variations according to time were observed with the wild-type <i>parS</i> (green curves) and the random <i>parS</i> (red curves). Various concentrations of crude extracts were injected: 10 (dotted lines), 20 (dashed lines) 40 μg/ml (full lines) for c1; 20 (dotted lines), 40 (dashed lines) and 80 μg/ml (full lines) for pBC, c3, 12D, 12J and G4.</p

Positions of two origins visualized simultaneously.

<p><b>A</b>. The origins of two replicons are plotted relative to the distance from the pole nearest any focus. Insets show numbers of cells with two foci of the replicon correspondingly coloured in the plot, comprising those with either one or two foci of the other replicon visualized; cells lacking foci of either replicon were excluded from the analysis. Red and green discs indicate respectively Chfp and Gfp fusions used to mark the origins shown. <b>B</b>. Examples of cells with origins of two replicons co-visualized. The separate components of the overlays are shown as examples of the images on which length measurements were made. Scale bar is 1 μm. <b>C</b>. Frequencies of focus combinations, shown as percentage of total cells scored.</p

Role of Par systems in cell growth and morphology.

<p><b>A</b>. Size of colonies formed at 3 days from cells transformed by plasmids carrying the <i>parS</i> sites indicated, either as single sites (e.g. Sc2) or as the natural clusters (e.g. Sc2+). <i>Bcen</i> makes pigment as cells enter stationary phase: faster-growing colonies are thus brown, slower-growing colonies still white. <b>B: left panels</b> Time of appearance of colonies after spreading on the agar media shown. Colonies were scored every 8 hours until the count reached its maximum; <b>middle panels</b> Cells grown exponentially in SOB were fixed, stained with DAPI and scored by fluorescence microscopy; <b>right panels</b> Viability on solid media. Cells grown in SOB to OD₆₀₀ ~1.0 were diluted and plated on LB agar either as a 0.1ml sample spread with glass beads or as a 10μl drop. The numbers of colonies are expressed as the ratio to the wild type colony count corrected for differences in OD. <b>C</b>. Cell dimensions. Cells growing exponentially in SOB at 37°C were fixed for viewing by phase contrast microscopy and their length (top panels) and width (bottom panels) were measured. The reference strains for the <i>ΔA(B)</i> mutants and <i>parS</i>-transformants were Nel13 without and with pMMBΔ respectively. <b>D</b>. Illustrative examples of abnormal cell phenotypes characteristic of mutants Δ<i>parA</i>c1 (top, and see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006172#pgen.1006172.s011" target="_blank">S8 Fig</a>) and Δ<i>parAB</i>c2 (bottom). The nucleoids of the Δ<i>parAB</i>c2 cells are revealed by DAPI staining. Scale bars are 2 μm.</p

Position of origins relative to cell poles.

<p><b>A</b>. Graphical summary of distances from the pole nearest a focus to the foci in one- and two-focus cells, and below, a plot of inter-focus distances. <b>B</b>. The positions of foci formed on <i>ori</i>-proximal sites by (<b>c1</b>) ParBc1::Gfp, (<b>c2</b>) ParBc2::Chfp, (<b>c3</b>) ParBP1::Gfp and (<b>p1</b>) ParB::Chfp were measured relative to the cell pole nearest a focus and plotted against cell length. Distances in cells with a single focus are shown as black-bordered circles in all cases; distances in cells with two foci are shown as coloured symbols. Arrows indicate the beginning of the two-focus clusters (left) and the end of the main one-focus clusters (right). Insets show numbers of cells scored, in corresponding colours. <b>C</b>. Examples of cells showing ParB::fp-marked origin regions. Scale bar is 1 μm.</p

Replication characteristics determined by high-throughput sequencing.

<p><b>A</b>. Base-pair frequency gradients. Data points are averages of binned read numbers representing successive blocks of 10kbp for c1 and c2 and 1kbp for c3. The top panels show the raw data for DNA from exponentially-growing cells, the third row shows the same data after division by the correspondingly binned data obtained from stationary-phase cells (second row). Nucleotide positions on the abscissa are reversed to conform to the intuitive sense of right and left chromosome arms. Because the data are plotted as raw read frequencies, relative copy numbers of the replicons can be read from the ordinates. <b>B</b>. Calculation of chromosome replication period, C, and speed. Origin/terminus ratios were used to calculate the time taken to replicate each chromosome arm from <i>ori</i>/<i>ter</i> = 2^C/τ. In the case of c1 and c2, the concavity of the stationary-phase base-pair frequency curves would falsify calculation of origin/terminus ratios from normalized data, necessitating use of the raw data plot. For this, raw data <i>ori</i>- and <i>ter</i>-proximal points that corresponded to points intersected by the linear regression plot of the normalized data were connected by lines whose upper extremities and intersection were taken as the <i>ori</i> and <i>ter</i> values respectively. The c3 stationary-phase bp frequency curve is essentially flat, validating the normalized data. The * values for c3 replication speed are calculated on the basis of terminus displacement creating arms estimated to be 340 and 530 kb long.</p

Replication characteristics of chromosomes in Δ<i>parA</i>c1 mutant cells.

<p><b>A</b>. DNA purified from exponentially-growing FBP47 (Nel13 Δ<i>parA</i>c1) cells was purified, processed and analyzed in parallel to wild type (Nel13) DNA, as outlined in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006172#pgen.1006172.g002" target="_blank">Fig 2</a> legend. The FBP47 data are shown in orange superimposed on the Nel13 data in blue (transposed from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006172#pgen.1006172.g002" target="_blank">Fig 2</a>). Raw read numbers are shown for c1 and c2: these reflect relative quantities of loci within a given strain but not of a given replicon between wt and mutant strains. <b>B</b>. Positions of c1 and c2 origins in wild type and Δ<i>Ac1</i> mutant cells growing in MglyC medium at 30°C. Pole-to-focus distances are shown black for one-focus cells and coloured for two-focus cells, as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006172#pgen.1006172.g003" target="_blank">Fig 3</a>. In these conditions, cells grow more slowly (τ ~140 mins) than in the previous analyses of focus position shown in Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006172#pgen.1006172.g003" target="_blank">3</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006172#pgen.1006172.g004" target="_blank">4</a> (τ ~110 mins), and the cells are correspondingly smaller.</p

Segregation and positioning of replication origin regions during the <i>Bcen</i> cell cycle.

<p>Sequential partition in the order c1 –c2 –c3 –p1 is depicted. All origins are shown as gravitating to the centre of cell halves before division, as deduced from their central position in single-focus cells.</p