ParB silencing test for wild type and mutated versions of <i>parS</i>.

<p><i>E</i>. <i>coli</i> DH5α transformants carrying pGB2 derivatives with individual wt <i>parS</i> sequences or their mutated versions were transformed with pKLB2 (<i>tacp-parB</i>_{<i>P</i>.<i>a</i>.}). Three independent transformation experiments were conducted and representative results are demonstrated. Undiluted transformation mixtures and their 10- and 100-fold dilutions were plated on three types of selection plates. Numbers of colonies growing on L-agar plates with Pn (blue bar), Pn and Sm (red bar), and double selection plates with 0.5 mM IPTG (green bar) are shown for the undiluted samples.</p

The <i>parS</i> sites and their localization in the <i>Pseudomonas aeruginosa</i> genome.

<p><b>(A)</b> Circular map of the <i>P</i>. <i>aeruginosa</i> genome with locations of putative ParB binding sequences [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120867#pone.0120867.ref009" target="_blank">9</a>]. Position of the <i>parAparB</i> operon is shown as black rectangle, grey arrow marks <i>oriC</i>, black arrows indicate predicted <i>parS</i> sites. <b>(B)</b> Nucleotide sequences, genomic coordinates and gene locations of the <i>parS</i> sites. The sequences are presented in a clockwise configuration. The coordinates are given according to the genomic sequence of the PAO1-UW strain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120867#pone.0120867.ref069" target="_blank">69</a>]. <b>(C)</b> Sequence logo for all twenty 8-bp half-sites in the <i>P</i>. <i>aeruginosa</i> PAO1-UW genome <b>Error! Bookmark not defined.</b>). Nucleotides at positions 2 and 5 are invariant in all half-sites.</p

Phenotypes of selected PAO1161 <i>parS</i> mutants.

<p>Colony morphology, swarming motility, intracellular ParB localization with the use of FITC-conjugated anti-ParB antibodies, and proportion of anucleate cells after DAPI staining are shown for representative mutant strains. As the controls wt PAO1161 Rif^R and <i>parA</i>_null and <i>parB</i>_null mutants were tested in each set of experiments. The percentage of anucleate cells is the mean from at least three independent experiments, with approximately 1000 cells counted in each experiment.</p

Summary of phenotypes of various <i>parS</i> mutants.

<p>The collection comprises 10 mutants with each individual <i>parS</i> modified (single) and 21 multiple mutants with between two and ten (<i>parS</i>_null) <i>parS</i> sequences modified (red dots). Mutants in columns highlighted in green demonstrate wild-type phenotypes (wt-like), those highlighted in yellow show phenotypes similar to those observed for the <i>parA</i>_null and <i>parB</i>_null mutants (<i>parAB</i>-like). The PAO1161 Rif^R<i>parS</i>mut29 mutant has ectopic <i>parS2</i> (green dot) replacing <i>parS7*</i> in <i>parS</i>_null mutant (highlighted in blue).</p

The hierarchy of ParB binding to parS sequences.

<p>Fluorescently labelled ds <i>parS</i> oligonucleotides (6 pmoles) were incubated with 240 pmoles of His₆-ParB and increasing amounts of unlabelled ds <i>parS2</i> as competitor: <b>(A)</b> 18 pmoles <b>(B)</b> 60 pmoles <b>(C)</b> 90 pmoles and <b>(D)</b> 180 pmoles. Complexes were visualized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120867#pone.0120867.g003" target="_blank">Fig. 3</a>.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Validation of the microarray data by RT-qPCR.

<p>Validation of the microarray data by RT-qPCR.</p

Bacterial strains used in this study.

<p>Bacterial strains used in this study.</p

Transcriptome changes in response to ParB overproduction.

<p>(<b>A</b>) Statistics of loci with significant expression change (FC<-2 or >2, <i>p</i>-value <0.05). RNA was isolated from PAO1161 cultures grown in L broth without Ara (WT), PAO1161 (pKGB8 <i>araBAD</i>p) cultures grown under selection in L broth with 0.02% Ara (empty vector control, EV), PAO1161 (pKGB9 <i>araBAD</i>p-<i>parB</i>) cultures grown under selection in L broth without Ara (mild ParB excess, ParB+) or with 0.02% Ara (higher ParB excess, ParB<i>+++</i>). (<b>B</b>) Venn diagram for sets of loci with significant expression change between EV <i>vs</i> WT, ParB+ <i>vs</i> EV and ParB+++ <i>vs</i> EV. (<b>C</b>) Classification of loci with altered expression according to PseudoCAP categories [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181726#pone.0181726.ref057" target="_blank">57</a>]. When a gene was assigned to multiple categories, one category was arbitrarily selected (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181726#pone.0181726.s006" target="_blank">S2 Table</a>). The PseudoCAP categories were grouped into six classes as marked. White and black bars correspond to the numbers of respectively, upregulated and downregulated genes in a particular category.</p

Influence of ParB on gene expression in the <i>parS1-4</i> region.

<p>(<b>A</b>) Mean level of expression of <i>dnaA</i>-<i>def</i> (<i>PA0001</i>-<i>PA0019</i>) genes in ParB+ and ParB+++ cells relative to EV as revealed by microarray analysis. Filled markers indicate statistically different expression relative to EV (<i>p</i>-value < 0.05 in ANOVA test). Arrangement of the genes in the chromosome is shown below. Operons (according to the DOOR 2.0 database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0181726#pone.0181726.ref073" target="_blank">73</a>]) are marked in grey. (<b>B</b>) Expression of <i>PA0001</i>-<i>PA0016</i> <b>(</b><i>dnaA</i>–<i>trkA</i>) genes in ParB+ and ParB+++ cells relative to EV cells. (<b>C</b>) RT-qPCR analysis of expression of <i>dnaA</i>–<i>trkA</i> genes in <i>parB</i>_null and <i>parS</i>_null strains relative to WT cells. Cells were grown in L broth. (<b>D</b>) RT-qPCR analysis of expression of <i>dnaA</i>–<i>trkA</i> genes in <i>parS</i>_null (pKGB9 <i>araBADp</i>-<i>parB</i>) and <i>parS</i>_null (pKGB8 <i>araBAD</i>p) relative to EV [PAO1161 (pKGB8 <i>araBAD</i>p)]. Cells were grown in L broth supplemented with chloramphenicol and 0.02% arabinose. RT-qPCR data represent mean ±SD from three biological replicates. Filled symbols indicate significantly different expression (<i>p</i>-value <0.05 in two-sided Student’s <i>t</i>-test assuming equal variance) relative to the control cells labelled as blue squares. The differences in expression of genes in <i>parS</i>_null (pKGB9) strain relative to <i>parS</i>_null (pKGB8) strain are not statistically significant.</p