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

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

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

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

Gene expression analysis in logarithmically growing cultures of <i>parA</i>_null, <i>parB</i>_null strains versus WT <i>P. aeruginosa</i>.

<p>(<b>A</b>) Quality analysis of three biological replicates of studied strains <i>parA</i>_null, <i>parB</i>_null and WT of PAO1161 <i>P. aeruginosa</i> by principle component analysis (PCA) of data obtained from expression microarray analysis. The first principle component (PC#1) accounted for 68% and the second principle component PC#2 for 19.7% of the total variation in the dataset. The plot indicates that the transcriptome data are of high quality as the samples cluster together according to the strain: green - WT, red - <i>parA</i>_null, blue - <i>parB</i>_null. (<b>B</b>) and (<b>C</b>) Cluster analysis of the normalized gene expression for genes that were differentially regulated in <i>parA</i>_null and <i>parB</i>_null strains as compared to the WT, respectively.</p

Number of genes with changed expression in <i>P. aeruginosa parA</i>_null and <i>parB</i>_null strains.

<p>The loci with altered expression in <i>parA</i>_null and <i>parB</i>_null as compared to reference PAO1161 <i>P. aeruginosa</i> (WT), indicated by pairwise comparison of microarray data (fold change FC ≥2; p-value ≤0.05). Number of genes (including intergenic regions and tRNA genes) with indicated mRNA level change are shown. Genes were grouped according to the magnitude of differential expression.</p

Functional classification of genes differentially expressed in logarithmically growing cultures of <i>P. aeruginosa par</i> mutants.

<p>(<b>A</b>) Venn diagram demonstrating the number of genes with changed mRNA level (fold change ≥2; p-value ≤0.05) in <i>parA</i>_null and <i>parB</i>_null mutant strains as compared to reference WT PAO1161 <i>P. aeruginosa</i> strain. Three gene set lists were created representing genes differentially expressed only in <i>parA</i>_null, with different expression in both <i>par</i> mutants (common in <i>parA</i>_null and <i>parB</i>_null) and with different mRNA level only in <i>parB</i>_null. (<b>B</b>) Functional classification of identified genes according to their predicted or known functions. Functional classes are taken from PseudoCAP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087276#pone.0087276-Winsor1" target="_blank">[29]</a> and are listed on the left with abbreviations in brackets. The original PseudoCAP functional categories were further grouped into six larger classes encompassing: (<b>I</b>) adaptation, protection, motility (green panel); (<b>II</b>) membrane proteins, transport, secretion (blue panel); (<b>III</b>) signal transduction, regulatory functions (red panel); (<b>IV</b>) cellular processes (yellow panel); (<b>V</b>) metabolism (orange panel); (<b>VI</b>) hypothetical, unknown functions (grey panel). (<b>C</b>) The pie charts created for each gene set list illustrating the percentage of genes in each class accounted for the total number of genes with changed expression for: only in <i>parA</i>_null, common in <i>parA</i>_null and <i>parB</i>_null and only in <i>parB</i>_null gene set list.</p

Validation of microarray data by RT-qPCR analysis.

<p>The RT-qPCR on RNA samples applied for microarrays analysis for chosen genes with changed mRNA level in <i>P. aeruginosa par</i> mutants (p-value ≤0.05; fold change ≥2). Abbreviations: MC - microarray data; RT - RT-qPCR data; SD RT - standard deviation for RT-qPCR analysis; nd - change not detected. Standard deviation from at least three independent experiments is presented.</p

Genes from overrepresented regulons affected by mutations in <i>parA</i> and <i>parB</i> genes in <i>P. aeruginosa</i>.

<p><b>(A)</b> RpoS, QS, RpoN(KinB) and stress regulated genes with altered expression in <i>par</i> mutants presented as Venn diagram illustrating separate and common genes classified into presented regulons (according to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087276#pone.0087276.s001" target="_blank">Table S1</a>). (<b>B)</b> The most potent known regulators with changed expression in <i>par</i> mutants. The functions they influence are given.</p